Lens-free holographic optical system for high sensitivity label-free microbial growth detection and quantification for screening, identification, and susceptibility testing

ABSTRACT

Disclosed are optical interrogation apparatus that can produce lens-free images using an optoelectronic sensor array to generate a holographic image of sample objects, such as microorganisms in a sample. Also disclosed are methods of detecting and/or identifying microorganisms in a biological sample, such as microorganisms present in low levels. Also disclosed are methods of using systems to detect microorganisms in a biological sample, such as microorganisms present in low levels. In addition or as an alternative, the methods of using systems may identify microorganisms present in a sample and/or determine antimicrobial susceptibility of such microorganisms.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of U.S. patent applicationSer. No. 17/353,943, filed Jun. 22, 2021, which is a continuation of,and claims priority to, U.S. patent application Ser. No. 16/028,287, nowU.S. Pat. No. 11,079,719, filed Jul. 5, 2018, which claims the benefitof U.S. Provisional Patent Application No. 62/528,825, filed Jul. 5,2017, the disclosures of which are hereby incorporated by reference intheir entirety.

FIELD

The present disclosure relates to cell and microorganism detection.

BACKGROUND

Microbial infections are best treated as early as possible to confer thegreatest opportunity for patient recovery and to limit morbidity andmortality. For example, roughly 85% of patients demonstrating symptomsof infection will not have sufficient microorganism concentrations intheir blood at initial presentation to enable detection of the causativeagent. Corresponding blood samples may appear negative formicroorganisms until many doubling events occur, at which pointsufficient numbers of microbial cells will be present and reach thelower threshold of standard detection testing.

Automated microscopy systems traditionally used to detect host andmicrobial cells (hereinto referred to as cells, microbes, organisms andmicroorganisms) in patient samples comprise various configurations ofsample containers, reaction reservoirs, reagents, and optical detectionsystems. Such optical detection systems are generally configured toobtain images via, for example, dark field and fluorescencephotomicrographs of microorganisms contained in reaction reservoirs,such as flowcells (e.g., microfluidic channels/chamber, perfusionchambers, and the like). Such optical detection systems also comprise acontroller configured to direct operation of the system and processmicroorganism information derived from photomicrographs. These systemsgenerally are not capable of detecting extremely low concentrations ofcells and microorganisms in direct from patient samples, and require aculturing period to ensure that if present, viable cells reach adetectable level to statistically ensure that a negative detectionreading is truly negative.

A phenotypical approach to detection of a viable cell or microbialpopulation in a sample involves in vivo monitoring of cell growth. Whilemany approaches have been proposed to achieve this (impedance, weight,growth by-product concentration monitoring, etc.), solutions based ondirect optical interrogation remain elusive as an alternative. Opticalapproaches are typically constrained by factors such as opticalresolution as well as the need for timely acquisition of cell division(growth) over time (time-lapse microscopy). For example, detection ofsmall concentrations of viable bacteria (typically <<10⁵ cfu/mL)presents additional challenges as it requires large volumes of directfrom patient sample to be interrogated (on the order of milliliters) toensure a high probability of detection. Moreover, achieving bettersensitivity in time-to-detection, such large volumes need to be scannedat rates higher than cellular division rates.

Usually, optical interrogation at high resolution relies on lengthymultiple pass scanning methodologies employing high precision 3-Dstages, high quality objectives, and fine focusing techniques. Moreover,label-free (unstained) cells require the employment of less commonimaging modes—such as phase contrast or differential contrastinterference microscopy—due to a very small difference in refractiveindex with suspension media. As a result, hardware and softwarerequirements for such applications scale poorly with sample volume underexamination.

SUMMARY

To address this problem of detecting the presence of cells and organismsin low concentrations in patient samples, an imaging system was devised,which uses a three-dimensional (“3D”) or four-dimensional (“4D”)holographic approach. For example, images of a patient sample in avolume (3D) can be obtained over time (4D), for example using videoframe rates. Unlike conventional imaging techniques, the instantholographic imaging system does not rely on multiple focal planes ofcells growing in a location requiring repeated image capturing overtime. Instead, a matrix array of optoelectronic sensors is employed toobtain a plethora of single images captured per time point from a 3D or4D suspension of cells and microorganisms in a medium whose propertiesphysically retain cells and microorganisms in a volume of sample and insome cases more substantially immobilized in a single location. As thosecells divide, their offspring remain in the proximity of each otheraffording rapid detection of localized growth or tracking of individualcell movement with observation frequent enough to track individualcells. Furthermore, in the case of immobilization, the offspring remainproximal to the location of their mother cells, eliminating the need totrack individual cell movement across a large volume of sample. In allembodiments, the focal point is numerically determined after theholographic image is captured affording significant advantages, one ofwhich being the elimination of motion in the z direction to captureimages at multiple focal points. The process permits simultaneousimaging of a large volume of patient sample to improve the chances ofdetecting viable cells present in low concentrations. Furthermore,because rate of acquisition of an entire 3D/4D volume of informationacross the matrix array of optoelectronic sensors is limited byelectronic readout time and essentially real-time or near real time onthe timescale of typical cell life cycle (on the order of millisecondscompared to average doubling rates of 2 to 3 divisions per hour withbacteria) the constraint on physical retention of cells in a singlelocation is significantly reduced affording flexibility to support adiverse set of requirements associated with a number of conventionaltests. The embodied holographic imaging system enables sufficientlyinstant microscopic examination of these methods conferring advantagesincluding but not limited to the near real time or instantaneousmonitoring of cells and microorganisms and the response to theenvironment during testing or characterization.

In one embodiment, the media can be essentially water-like liquid brothgrowth media, or a desired minimal media, or a suspended biologicalsample (such as a blood, urine, respiratory, or saliva sample). Liquidgrowth media conditions are relevant to diagnostic testing including butnot limited to traditional culturing methods to detect cell presence indiagnostic samples and susceptibility testing like Broth MicrobiologyDilution (BMD) testing (CLSI. Methods for Dilution AntimicrobialSusceptibility Tests for Bacteria that Grow Aerobically, 1 edition. CLSIguideline M07-ED11. Wayne, Pa.: Clinical and Laboratory StandardsInstitute; 2018. The entire contents of this reference incorporatedwithin this application).

In another embodiment involving bacteria, the retention of themicroorganisms can be achieved using conventional manual or automatedprocesses that deposit of organisms on agar plates (including but notlimed to manual streaking of plates using swabs or automated means toaccomplish the same) in a substantially uniform layer, as required withconventional Kirby Bauer or Disk Diffusion methods (CLSI. PerformanceStandards for Antimicrobial Disk Susceptibility Tests, 13th edition.CLSI guideline M02-ED11. Wayne, Pa.: Clinical and Laboratory StandardsInstitute; 2018. The entire contents of this reference incorporatedwithin this application),

In yet another embodiment, droplet deposition of liquid suspensions oforganisms on agar plates as described in traditional plating andovernight culture of micro-organisms (L. S. Garcia (ed.), 2007 Update:Clinical Microbiology Procedures Handbook, 2nd ed., 2007 Chapter 3,Section 11.2 Lower Respiratory Tract Cultures) and the agar dilutionmethod of susceptibility testing (CLSI. Methods for DilutionAntimicrobial Susceptibility Tests for Bacteria that Grow Aerobically,11th edition. CLSI guideline M07-ED11. Wayne, Pa.: Clinical andLaboratory Standards Institute; 2018. The entire contents of thisreference incorporated within this application).

Further embodiments, include but are not limited to, use of agar overlayor similar methodologies that enhance immobilization of organismsdepending on the requirements of the testing performed. Provided hereinis a system that includes a holographic optical apparatus situated todetermine the presence of a cell or microorganism in a sample volumebased on a detected variation over time of a hologram of the samplevolume, such as a detected variation corresponding to four or fewer celldoubling events, or three or fewer cell doubling events. In someexamples, the holographic apparatus is an in-line holographic apparatus,and the hologram is an in-line hologram. In some examples, the in-lineholographic optical apparatus includes a reference beam source situatedto direct a reference beam to the sample volume; a sample receptaclesituated to hold the sample volume in view of the reference beam; anoptical sensor (such as a complementary metal oxide semiconductor (CMOSor CCD) sensor having a pixel pitch of 1.5 μm or smaller) situated todetect the in-line hologram formed by the reference beam and the samplevolume; and a controller coupled to the optical sensor and configured todetermine the variation over time of the in-line hologram. In someexamples, the optical sensor has a pixel pitch of 1 μm/pixel or smallerand the controller is configured to determine, based on the detectedin-line hologram, morphological characteristics of the microorganismdetermined to be present. The reference beam source can include apinhole aperture situated to receive multi-wavelength illumination froman illumination source and the reference beam is directed lens-free fromthe pinhole aperture to the sample volume and optical sensor. In someexamples, the reference beam source is situated to direct a plurality ofreference beams to the sample volume and to adjacent portions of theoptical sensor so as to mosaic the field of view of the in-lineholographic apparatus, for example, wherein the adjacent portions of theoptical sensor portions correspond to separate CMOS sensors. In someexamples, the multi-wavelength illumination received by the illuminationsource is incoherent and the reference beam comprises incoherentillumination. In some examples, the controller is configured toreconstruct the spatial characteristics of the sample volume based onthe detected in-line hologram, diffraction propagation approximation,and a phase retrieval algorithm. In some examples, the controller isconfigured to determine a focal plane of the cell or microorganism inthe sample volume. In some examples, the sample volume includes at leastone sample reaction chamber situated as a growth control with a firstsample portion situated in the absence of an antimicrobial agent, and atleast one sample reaction chamber situated as an antimicrobialsusceptibility test with a second sample portion situated in thepresence of an antimicrobial agent. The sample volume can include aplurality of growth channels having selective media. In some examples,the holographic apparatus is situated to determine the presence based onthe detected variation with the sample volume having a microorganismconcentration of 100 CFU/mL or less, such as 10 CFU/mL or less. In someexamples, the holographic apparatus is situated to display a time-lapseimage associated with the sample volume at a time-resolution that isfaster than a microorganism division rate. In some examples, thetime-lapse image corresponds to one or more of the hologram and one ormore planes of the sample volume.

Also provided are methods for detecting a microorganism in a sample (andin some examples also identifying the microorganism, determining theantimicrobial susceptibility of the microorganism, or both), which canuse the disclosed systems (such as those that utilize holography). Insome examples, the sample is a polymicrobial sample. In some examples,the biological sample comprises 100 CFU/mL or less, such as 10 CFU/mL orless, of the microorganism. In some examples, the microorganismcomprises bacteria, protozoa, fungi, or combinations thereof. In someexamples, the method includes detecting an in-line hologram of asuspended biological sample (such as a blood, urine, respiratory, orsaliva sample); and for at least one object in the suspended biologicalsample, determining a variation over time of the in-line hologram thatis associated with an indication that the at least one object is a cellor microorganism in the biological sample. In some examples, determininga variation over time includes determining a spatial difference overtime associated with the at least one object and corresponding to acell's growth or decline. In some examples, the suspended biologicalsample is suspended in a porous medium, and the method further includesincubating the suspended biological sample in an environment conduciveto cell replication (e.g., growth, division, or both). In some examples,the method further includes interrogating the suspended biologicalsample in an optical interrogation system; wherein the opticalinterrogation system includes at least one optical sensor situated toperform the detecting of the in-line hologram. In some examples, themethod further includes determining a focal plane corresponding to aplane of highest variance in the suspended biological sample that isassociated with the at least one object. In some examples, the methodfurther includes reconstructing spatial characteristics of the suspendedbiological sample based on the detected in-line hologram and a numericalreconstruction algorithm. In some examples, the optical sensor has apixel pitch of 1 μm/pixel or smaller and the method further includesdetermining, based on the detected in-line hologram, morphologicalcharacteristics of the at least one object corresponding to amicroorganism. In some examples, the method further includes directing aplurality of reference beams to the suspended biological sample and toadjacent portions of the optical sensor so as to mosaic the field ofview of the optical interrogation system. In some examples, thesuspended biological sample includes at least one sample reactionchamber situated as a growth control with a first sample portionsituated in the absence of an antimicrobial agent, and at least onesample reaction chamber situated as an antimicrobial susceptibility test(AST) with a second sample portion situated in the presence of at leastone antimicrobial agent. An exemplary growth control includesMueller-Hinton media in broth formulation (MHB) or agar formulation(MHA) that enables immobilization or entombment for immobilizationpurposes. Exemplary antimicrobial agents include one or more ofamikacin, ampicillin, ampicillin-sulbactam, aztreonam, cefazolin,cefepime, ceftaroline, ceftazidime, ceftriaxone, ciprofloxacin,colistin, daptomycin, doxycycline, erythromycin, ertapenem, gentamicin,imipenem, linezolid, meropenem, minocycline, piperacillin-tazobactam,tobramycin, trimethoprim-sulfamethoxazole, and vancomycin. The suspendedbiological sample can be present in a plurality of flow cells orchambers, each comprising selective and differential media, such asblood containing broth or agar, Eosin Methylene Blue (EMB) broth or EMBagar, mannitol salt) broth or mannitol salt agar, MacConkey) broth orMacConkey agar, phenylethyl alcohol (PEA)) broth or PEA agar, and YM)broth or YM agar, by way of example and not limitation. In someexamples, the method further includes displaying a time-lapse imageassociated with the suspended biological sample at a time-resolutionthat is faster than cell division or multiplication rate (e.g., the rateat which a bacterium or yeast divides into two daughter cells, the rateat which a protist divides itself into two or more daughter cells). Insome examples, the time-lapse image corresponds to one or more of thedetected in-line hologram and one or more planes of the suspendedbiological sample.

In some examples, the methods include detecting a variation of anin-line hologram over time of a biological sample; and determining thepresence of a cell or microorganism in the biological sample based onthe detected variation.

In some examples, the methods include detecting an in-line hologram of abiological sample at a first time and a second time; comparing thein-line holograms to determine a hologram variation associated with thecell or microorganism; and determining whether a microorganism ispresent in the biological sample based on the variation.

In some examples, the system includes at least one processor, and one ormore computer-readable storage media including stored instructions that,responsive to execution by the at least one processor, cause the systemto compare a first in-line hologram of a sample volume at a first timeand a second in-line hologram of the sample volume at a second time andto determine a hologram variation between the first in-line hologram andsecond in-line hologram that is associated with an indication as to thepresence of a cell or microorganism in the sample volume.

In particular sequences of using holographic optical apparatus andmethods examples herein, screening can be performed to determine thepresence of a microorganism, AST can be performed, and thenidentification.

Also provided are optical interrogation platform systems. In someexamples, such a system includes an in-line holographic setup comprisinga single-aperture multi-wavelength illumination; and a complementarymetal oxide semiconductor (CMOS) sensor having a pixel pitch selected soas to detect a holographic variation over time associated with thepresence of a cell or microorganism in a sample volume.

Also provided are automated methods of lens-free microscopy fordetecting one or more cells or microorganisms in a sample (and in someexamples also identifying the microorganism, determining theantimicrobial susceptibility of the microorganism, or both). In someexamples, the methods include suspending a biological sample in a porousmedium; introducing the suspended biological sample to a sample reactionchamber; subjecting the porous medium to a phase change to immobilizemicroorganism cells in the suspended biological sample inthree-dimensional space; incubating the suspended biological sample inan environment conducive to microorganism replication; interrogating thesuspended biological sample in an automated optical interrogation systemusing one or more optoelectronic sensors to locate the optimal focalplane for each microorganism in the sample; tracking spatial differencesto detect changes in growth of microorganisms over time; and acquiringholographic images of replicating microorganisms, thereby detectingtheir presence in the biological sample. In some examples, the phasechange produces a gelled medium. In some examples, the microorganismsare present in the biological sample at a concentration of approximately10² bacteria per 1 mL or 10² bacteria per 300 uL of sample.

According to another aspect of the disclosed technology, systems can beautomated and include an automated holographic optical apparatussituated to determine at least the antimicrobial susceptibility of amicroorganism corresponding to an object in a sample volume based on adetected variation over time of a hologram of the sample volume, anoutput of at least one deeply supervised convolutional neural network,and a phenotypical behavior of the microorganism, wherein thephenotypical behavior of the microorganism is classified based on thedetected variation and the output of the at least one deeply supervisedconvolutional neural network. In representative systems, the holographicapparatus is an in-line holographic apparatus and the hologram is anin-line hologram, and the in-line holographic optical apparatus includesa reference beam source situated to direct a reference beam to thesample volume, a sample receptacle situated to hold the sample volume inview of the reference beam, an optical sensor situated to detect thein-line hologram formed by the reference beam and the sample volume, anda controller coupled to the optical sensor and that includes at leastone processor and one or more computer-readable storage media includingstored instructions that, responsive to execution by the at least oneprocessor, cause the controller to determine the variation over time ofthe in-line hologram. In some examples, the controller is configured toreconstruct the spatial characteristics of the sample volume based onthe detected in-line hologram, diffraction propagation approximation,and a phase retrieval algorithm. In further examples, the controller isconfigured to determine a focal plane of the microorganism in the samplevolume based on the reconstructed spatial characteristics. In particularexamples, the at least one deeply supervised convolutional neuralnetwork includes a spatial reconstruction deeply supervisedconvolutional neural network configured to produce an outputcorresponding to a reconstruction of the spatial characteristics of thesample volume based on a trained set of network layers, and wherein thecontroller is configured to reconstruct the spatial characteristics ofthe sample volume using the reconstruction deeply supervisedconvolutional neural network. In selected examples, the at least onedeeply supervised convolutional neural network includes a microorganismidentification deeply supervised convolutional neural network configuredto produce an output corresponding to a microorganism identification,microorganism morphology identification, microorganism movementidentification, and/or microorganism phenotypic classification for themicroorganism in the sample volume based on a trained set of networklayers, and wherein the controller is configured to identify themicroorganism, microorganism morphology, microorganism movement, and/orclassify the microorganism phenotypical behavior using the microorganismidentification deeply supervised convolutional neural network. In someexamples, the controller is configured to determine a 3D position and/ormorphological characteristics of the microorganism based on the in-linehologram. In further embodiments, the controller is configured toassociate the object detected in a later hologram with the objectdetected in an earlier hologram, based on proximity or morphologicalcharacteristics of the objects detected from the variation over time ofthe in-line hologram. In particular examples, the controller isconfigured to form an object track for the object in the sample volumebased on the detected variation over time of the in-line hologram. Insome embodiments, the controller is configured to identify the object asthe microorganism in the sample volume based on the detected variationover time of the in-line hologram. In some embodiments, the controlleris configured to classify a phenotypical behavior of the microorganismin the sample volume based on the detected in-line hologram. In someexamples classifying a phenotypical behavior, the controller isconfigured to determine a correspondence between the phenotypic behaviorof the microorganism and presence, concentration, and taxon of themicroorganism in the sample volume. In further examples classifying aphenotypical behavior, the sample volume includes a plurality of samplevolume portions situated in a respective at least one growth control, atleast one selective media, and at least one antimicrobial flow cell thatare held by the sample receptacle, and the controller is configured todetermine the presence, taxon, and an antibiogram of the microorganismor multiple microorganisms based on the at least one growth control, theat least one selective media, and the at least one anti-microbial flowcell. In some embodiments with an optical sensor, the optical sensor isa complementary metal oxide semiconductor (CMOS) sensor having a pixelpitch of 1.5 μm or smaller. In further embodiments with an opticalsensor, the optical sensor has a pixel pitch of 1 μm/pixel or smallerand the controller is configured to determine, based on the detectedin-line hologram, morphological characteristics of the microorganism. Insome embodiments, the reference beam source includes a plurality ofpinhole apertures spaced apart from each other by 1 mm or less with eachof the pinhole apertures configured to emit respective referencesubbeams at different respective wavelengths. In further embodiments,the reference beam source includes a pinhole aperture situated toreceive illumination from an illumination source and the reference beamsource is configured to direct the reference beam lens-free from thepinhole aperture to the sample volume and optical sensor. In somepinhole aperture examples, the illumination source is configured togenerate illumination at multiple wavelengths. In further pinholeaperture examples, the illumination received from the illuminationsource by the pinhole aperture is incoherent and the reference beamcomprises incoherent illumination. In some embodiments, the referencebeam source is situated to direct a plurality of reference beams to thesample volume and to adjacent portions of the optical sensor so as tomosaic the field of view of the in-line holographic apparatus. In somemosaic examples, the adjacent portions of the optical sensor correspondto separate CMOS sensors. In further examples, the sample volumeincludes a plurality of sample volume portions, including a first samplevolume portion situated in a first sample reaction chamber that is heldby the sample receptacle, wherein the first sample volume portion issituated as a growth control volume by having an absence of anantimicrobial agent, and including a second sample volume portionsituated in a second sample reaction chamber, wherein the second volumeportion is situated as an antimicrobial susceptibility test volume inthe presence of a predetermined antimicrobial agent. In particularexamples, the sample reaction chambers include a plurality of growthchannels having selective media. In some embodiments, the holographicapparatus is situated to determine a presence of the microorganism basedon the detected variation with the sample volume having a microorganismconcentration of 10 cfu/mL or less. In further embodiments, theholographic apparatus is situated to display a time-lapse imageassociated with the sample volume at a time-resolution that is fasterthan a microorganism division rate. In some time-lapse examples, thetime-lapse image corresponds to one or more of the hologram and one ormore planes of the sample volume. In some embodiments, a time period ofthe detected variation corresponds to four or fewer microorganismdoubling events. In further embodiments, a time period of the detectedvariation corresponds to three or fewer microorganism doubling events.In representative systems, the microorganism is in the sample volume.

According to a further aspect of the disclosed technology, methodsincludes detecting an in-line hologram of a suspended biological sample,measuring for at least one microorganism in the suspended biologicalsample, a variation over time of the in-line hologram, and determiningthe presence or absence of antimicrobial susceptibility for the at leastone microorganism in the suspended biological sample based on themeasured variation over time of the in-line hologram of the suspendedbiological sample, an output of at least one deeply supervisedconvolutional neural network associated with the measured hologram, anda phenotypical behavior of the at least one microorganism, wherein thephenotypical behavior is classified based on the detected variation andthe output of the at least one deeply supervised convolutional neuralnetwork. In some examples, the at least one microorganism is in thesuspended biological sample. Some embodiments can include, beforedetermining presence or absence of antimicrobial susceptibility,determining whether a microorganism is present in the suspendedbiological sample based on the measured variation over time of thein-line hologram. Particular examples include suspending a biologicalsample in a porous medium to form the suspended biological sample,introducing the suspended biological sample to a sample reactionchamber, subjecting the porous medium to a phase change to immobilizethe at least one microorganism in the suspended biological sample inthree-dimensional space, incubating the suspended biological sample inan environment conducive to microorganism replication, wherein detectingthe in-line hologram and determining the variation over time includesinterrogating the suspended biological sample in an automated opticalinterrogation system using one or more optoelectronic sensors to locatean optimal focal plane for each of the microorganisms in the biologicalsample, tracking spatial differences to detect changes in growth of theat least one microorganism over time, and acquiring holographic imagesof the replicating at least one microorganism, thereby detecting itspresence in the biological sample. In some examples, the phase changeproduces a gelled medium. In further examples, the at least onemicroorganism is present in the biological sample at a concentration ofapproximately 10² bacteria per 1 mL of sample. In some examples, the atleast one microorganism is, and the determining a variation over timeincludes determining a spatial difference over time associated with theat least one microorganism and corresponding to a microorganism growthor decline. In selected examples, the at least one deeply supervisedconvolutional neural network includes a spatial reconstruction deeplysupervised convolutional neural network configured to produce an outputcorresponding to a reconstruction of the spatial characteristics of thesuspended biological volume based on a trained set of network layers. Inadditional examples, the at least one deeply supervised convolutionalneural network includes a microorganism identification deeply supervisedconvolutional neural network configured to produce an outputcorresponding to a microorganism identification, microorganismmorphology identification, microorganism movement identification, and/ormicroorganism phenotypic classification for the at least onemicroorganism in the suspended biological sample based on a trained setof network layers. In examples, the sample material of the suspendedbiological sample is suspended in a porous medium, and the suspendedbiological sample is incubated in an environment conducive tomicroorganism replication. In representative embodiments, the in-linehologram is detected with an optical sensor comprising one or moresensor portions. In selected examples, each of the optical sensorportions includes a plurality of pixels with a pixel pitch of 1 μm/pixelor smaller. Some examples can include directing a plurality of referencebeams to the suspended biological sample and to adjacent portions of theoptical sensor corresponding to the respective optical sensor portionsto produce a mosaicked field of view of the in-line hologram. Someembodiments include determining a focal plane corresponding to a planeof highest variance in the suspended biological sample that isassociated with the at least one object. Additional examples includereconstructing spatial characteristics of the suspended biologicalsample based on the detected in-line hologram and a numericalreconstruction algorithm. In some embodiments, the suspended biologicalsample is supported by a sample receptacle of an in-line holographyapparatus situated to perform the detecting, measuring, and determining,and wherein a first sample portion of the suspended biological sample islocated in a first sample reaction chamber in the absence of anantimicrobial agent so as to correspond to a growth control, and whereina second sample portion of the suspended biological sample is located ina second sample reaction chamber in the presence of at least oneantimicrobial agent. In some examples, growth control comprisesMueller-Hinton broth (MHB) or agar (MHA). In selected examples, the atleast one antimicrobial agent comprises amikacin, ampicillin,ampicillin-sulbactam, aztreonam, cefazolin, cefepime, ceftaroline,ceftazidime, ceftriaxone, ciprofloxacin, colistin, daptomycin,doxycycline, erythromycin, ertapenem, gentamicin, imipenem, linezolid,meropenem, minocycline, piperacillin-tazobactam, tobramycin,trimethoprim-sulfamethoxazole, vancomycin, or combinations thereof. Insome embodiments, suspended biological sample includes sample volumeportions that are present in a plurality of respective flowcellscomprising selective and differential media. In particular examples, theselective and differential media comprise blood broth or agar, EosinMethylene Blue (EMB) broth or EMB agar, mannitol salt broth or mannitolsalt agar, MacConkey broth or MacConkey agar, phenylethyl alcohol (PEA)broth or PEA agar, or YM broth or YM agar. Some embodiments can includedisplaying a time-lapse image associated with the suspended biologicalsample at a time-resolution that is faster than a microorganism divisionrate. In some time-lapse examples, the time-lapse image corresponds toone or more of the detected in-line hologram and one or more planes ofthe suspended biological sample. In some examples, the suspendedbiological sample is obtained from blood, urine, respiratory sample, orsaliva. In further examples, the suspended biological sample is apolymicrobial sample. In some examples, the suspended biological samplecomprises 10 CFU/ml or less of the at least one microorganism. Infurther examples, the microorganism comprises one or more bacteria,protozoa, fungi, or combinations thereof.

The foregoing and other objects and features of the disclosure willbecome more apparent from the following detailed description, whichproceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a lens free imaging using an optoelectronic sensor arrayto generate a holographic image of sample objects.

FIG. 2A is a view of a perfusion chamber mounted on a glass microscopeslide.

FIG. 2B is a view of alternative perfusion chambers with multipleindividual chambers, which can be mounted on a glass microscope slide,for example to analyze multiple samples contemporaneously, a singlesample under multiple different media, or combinations thereof.

FIG. 3 shows images obtained showing proof of concept of the opticalinterrogation platform using transparent silicone beads.

FIG. 4 shows images obtained by the optical interrogation platformimaging E. coli growth over a period of 0 to 180 minutes.

FIG. 5 shows images obtained by the optical interrogation platformimaging E. coli growth during a period from 240 to 540 minutes.

FIG. 6 is a perspective schematic of an example in-line holographicapparatus.

FIG. 7 is a perspective schematic of an example mosaicked in-lineholographic apparatus.

FIGS. 8-12 are flowcharts of example holography methods.

FIG. 13 is a schematic of an example computing environment.

FIGS. 14A-14C are perspective schematics of example sample volumesundergoing growth and detection with holography methods herein.

FIG. 15 is a flowchart of another example holography method.

FIGS. 16-17 are flowcharts of example convolutional neural networktraining and trained testing.

DETAILED DESCRIPTION

The following explanations of terms and methods are provided to betterdescribe the present disclosure and to guide those of ordinary skill inthe art in the practice of the present disclosure. The singular forms“a,” “an,” and “the” refer to one or more than one, unless the contextclearly dictates otherwise. For example, the term “comprising abacterium” includes single or plural bacteria and is consideredequivalent to the phrase “comprising at least one bacterium.” The term“or” refers to a single element of stated alternative elements or acombination of two or more elements, unless the context clearlyindicates otherwise. As used herein, “comprises” means “includes.” Thus,“comprising A or B,” means “including A, B, or A and B,” withoutexcluding additional elements. All references cited herein areincorporated by reference.

Unless explained otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood to one of ordinaryskill in the art to which this disclosure belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, suitable methods andmaterials are described below. The materials, methods, and examples areillustrative only and not intended to be limiting. For example, thesteps recited in any of the method or process descriptions may beexecuted in any order and are not necessarily limited to the orderpresented. Also, any reference to attached, fixed, connected or the likemay include permanent, removable, temporary, partial, full and/or anyother possible attachment option. Additionally, any reference to withoutcontact (or similar phrases) may also include reduced contact or minimalcontact. Furthermore, the connecting lines shown in the various figurescontained herein are intended to represent exemplary functionalrelationships and/or physical couplings between the various elements. Itshould be noted that alternative or additional functional relationshipsor physical connections may be present in a practical system. However,the benefits, advantages, solutions to problems, and any elements thatmay cause any benefit, advantage, or solution to occur or become morepronounced are not to be construed as critical, required, or essentialfeatures or elements of the inventions.

The disclosed methods, apparatus, and systems should not be construed aslimiting. Instead, the present disclosure is directed toward all noveland nonobvious features and aspects of the various disclosedembodiments, alone and in various combinations and subcombinations withone another. The disclosed methods, apparatus, and systems are notlimited to any specific aspect or feature or combination thereof, nor dothe disclosed embodiments require that any one or more specificadvantages be present or problems be solved.

In the detailed description herein, references to “one embodiment,” “anembodiment,” “an example embodiment,” etc., indicate that the embodimentdescribed may include a particular feature, structure, orcharacteristic, but every embodiment may not necessarily include theparticular feature, structure, or characteristic. Moreover, such phrasesare not necessarily referring to the same embodiment. Further, when aparticular feature, structure, or characteristic is described inconnection with an embodiment, it is submitted that it is within theknowledge of one skilled in the art to affect such feature, structure,or characteristic in connection with other embodiments whether or notexplicitly described. After reading the description, it will be apparentto one skilled in the relevant art(s) how to implement the disclosure inalternative embodiments.

Furthermore, no element, component, or method step in the presentdisclosure is intended to be dedicated to the public regardless ofwhether the element, component, or method step is explicitly recited inthe claims. No claim element herein is to be construed under theprovisions of 35 U.S.C. 112(f), unless the element is expressly recitedusing the phrase “means for.” As used herein, the terms “comprises,”“comprising,” or any other variation thereof, are intended to cover anon-exclusive inclusion, such that a process, method, article, orapparatus that comprises a list of elements does not include only thoseelements but may include other elements not expressly listed or inherentto such process, method, article, or apparatus.

In some examples herein, optical beam cross-sectional areas, diameters,or other beam dimensions can be described using boundaries thatgenerally correspond to a zero intensity value, a 1/e value, a 1/e²value, a full-width half-maximum (FWHM) value, or other suitable metric.As used herein, optical illumination refers to electromagnetic radiationat wavelengths of between about 100 nm and 10 μm, and typically betweenabout 200 nm and 2 μm. Optical illumination can be provided atparticular wavelengths (typically narrow wavelength bands) or ranges ofwavelengths.

As used herein, “AST” is antimicrobial susceptibility testing,antimicrobial agent susceptibility testing, or antibiotic susceptibilitytesting, and can include MIC (minimum inhibitory concentration) and/orSIR (susceptible, intermediate, resistant).

As used herein, “ID” is identification, such as a process of determiningthe species identity of a microorganism, such as determining oridentifying the genus, species, Gram status, and/or strain of amicroorganism. This is distinct from detecting the presence of anunknown microorganism in that it is more specific.

As used herein, “MHB” is Mueller Hinton Broth and MHA is Mueller HintonAgar.

As used herein, “3D” refers to three-dimensional space.

As used herein, “4D” refers to four-dimensional space.

In order to facilitate review of the various embodiments of thedisclosure, the following explanations of specific terms are provided:

Administration: To provide or give a subject an agent, such as anantimicrobial agent (such as an antibiotic or antifungal), by anyeffective route. Exemplary routes of administration include, but are notlimited to, oral, injection (such as subcutaneous, intramuscular,intradermal, intraperitoneal, intravenous, intra-articular, andintrathecal), sublingual, rectal, transdermal, intranasal, vaginal andinhalation routes.

Cell, microorganism or microbe: A microscopic cell or organism that insome examples causes disease, for example in a mammal, bird, or fish.Examples of cells include host cells in a host clinical specimen ormicroorganisms including, but not limited to bacteria, fungi (includingmold and yeast morphologies), and protozoa.

Sample or specimen: A biological sample or biological specimen, such asthose obtained from a subject (such as a human or other mammaliansubject, such as a veterinary subjects, for example a subject known orsuspected of having a disease or infection). The sample can be collectedor obtained using methods well known to those skilled in the art.Samples can contain nucleic acid molecules (such as DNA, cDNA, and RNA),proteins, cells, cell membranes, or combinations thereof. In someexamples, the disclosed methods include obtaining the sample from asubject prior to analysis of the sample using the disclosed methods anddevices. In some examples, a sample to be analyzed is lysed, extracted,concentrated, diluted, or combinations thereof, prior analysis with thedisclosed methods and devices.

Exemplary samples include, without limitation, cells, cell lysates,blood smears, cytocentrifuge preparations, flow-sorted or otherwiseselected cell populations, cytology smears, bodily fluids (e.g., bloodand fractions thereof such as white blood cells, serum or plasma;saliva; respiratory samples, such as sputum or lavages; urine;cerebrospinal fluid; gastric fluid; sweat; semen; puss; etc.), buccalcells; extracts of tissues, cells or organs, tissue biopsies (e.g.,tumor or lymph node biopsies); liquid biopsies; fine-needle aspirates;bronchoscopic lavage; punch biopsies; bone marrow; amniocentesissamples; autopsy material; fresh tissue; vaginal swabs; rectal swabs;and the like. The biological sample may also be a laboratory researchsample such as a cell culture sample or supernatant. As used herein,samples can include a sample volume and can be introduced to a containeror receptacle that houses or supports the sample volume. Sample volumescan include liquid or particulates of the sample (e.g., microorganisms,if present) obtained from a sampled subject. In typical examples, theliquid and/or particulate portions of the sample can include a mixturewith supporting media, such as growth media. The container or receptaclehousing the sample can include sample reaction chambers, which caninclude solid supports (e.g., polycarbonate, silicone, glass, etc.) intowhich patient sample material is loaded and which can define separationsbetween portions of the sample volume. In typical examples, “sample” canrefer to the material of a biological sample, such as when the materialis transferred between supporting structures (e.g., introducing a sampleto a flow cell). Sample receptacles can also refer to structures thatreceive and support samples and also structures that receive and supportsample containers that house samples.

Subject: Any mammal, such as humans and veterinary subjects, such as,non-human primates, pigs, sheep, cows, dogs, cats, rodents and the like.In one example, a subject is a human subject. In some examples, thesubject is known or suspected of having a disease or an infection. Insome examples, the subject is septic.

Overview

Patient samples, such as blood, respiratory, and other biologicalsamples, are the primary biological starting point for assessing theetiology of a patient's disease and determining the appropriate therapycourse for treating that disease. Key to reducing morbidity andmortality is initiating the proper therapeutic treatment of a criticallyill patient at the appropriate dosage regimen as soon as possible. Thehistorically weak link in this process is sufficient cultivation of acell or microbial population in the patient sample to enableidentification of pathogen(s) present and to determine which compoundsthe pathogen(s) will respond to in therapy. Reducing the assay timerequired to properly identify cells and microorganism(s) in a patientsample and assess their drug sensitivity is crucial to improving patientsurvival odds.

In many instances, patient samples contain only a single type ofmicroorganism. In other instances, patient samples contain multipletypes of cells microorganisms, such as mixtures of host cells andbacteria from differing genera, species, and even strains (also known as“polymicrobial” samples). Diagnostic accuracy is traditionally expressedin terms of sensitivity and specificity. Sensitivity refers to theprobability of assigning a diagnostic test as positive when it is infact, positive (the fraction of true positives), which confound theidentification and antimicrobial sensitivity processes. The counter tosensitivity is specificity, which is the rate of obtaining falsenegative test results. Current methods of identifying unknown cells ormicroorganisms are prone to failure in both false positive and falsenegative modes. These difficulties with sensitivity and specificity aretypically fostered by factors that impede sample detection, such asnoise, crosstalk, borderline resistance, and the like. Traditionalanalysis methods often trade sensitivity of detection for thespecificity of cell or microorganism identification. In otherapplications, the reverse is true, prioritizing sensitivity overaccurate identification. But to maximize efficiency, and thus improvethe odds of achieving a better treatment outcome for the patient,improving sensitivity for detecting the presence of cells as early aspossible is desirable. In doing so, clinicians and laboratory personnelcan determine which samples may be eliminated from the microbialidentification and antimicrobial sensitivity workflow stream due to atrue negative reading at the earliest possible time.

Traditional methods for identification (ID) and antimicrobialsusceptibility testing (AST) of organisms from clinical specimenstypically require overnight subculturing to isolate individual species(e.g., determine if the sample is positive for the presence ofpathogenic bacteria, protozoa, and/or fungi) prior to biochemicalassay-based identification, followed by growing isolated organisms inthe presence of various antimicrobials to determine susceptibilities.Although molecular identification methods can provide organismidentification in a few hours directly from clinical specimens as wellas resistance marker detection, these methods do not provide theantimicrobial susceptibility information required by clinicians toinform treatment decisions. Studies demonstrating the feasibility ofusing various sample types including whole blood and respiratory sampleshave been reported, but sample preparation techniques require furtherrefinement. Current rapid molecular-based diagnostic methods only reportidentification and genotypic resistance marker results. While availablein a couple of hours, these results only provide a partial answer. Thisleaves the clinician to prescribe overly-broad spectrum empiric therapywhile waiting two to four days for conventional antibioticsusceptibility test results before adjusting therapy. The availabilityof an antimicrobial susceptibility test result in as few as 5 hours orless, as opposed to a few days, potentially decreases morbidity andmortality in critically ill patients due to delays in administration ofappropriate therapy. In addition, rapid de-escalation frombroad-spectrum empiric therapies to targeted specific antimicrobialscould assist antimicrobial stewardship efforts to decrease the emergenceand spread of multi-drug resistant organisms (MDROs). By using thedisclosed holographic approach to determine which patient samplesactually have microorganisms present therein (e.g., as an alternative toovernight culturing), patients who can truly benefit from identificationand antimicrobial susceptibility testing can be pinpointed. Only thosepatients samples deemed positive for the presence of microorganismswould then be subjected to ID and AST evaluation, saving resources andtime. Furthermore, in some examples, microorganisms can be preciselyquantified, movements tracked, morphological characteristics identified,and/or phenotypic behavior classified.

To address these problems, the disclosed system provides an automatedmicroscopy system designed to provide rapid microorganism detectionprior to typical identification and antibiotic susceptibility testingresults. An aspect of this system is an optical interrogation platformcapable of detecting bacterial and/or fungal growth in a sample obtaineddirectly from a patient without prior overnight culturing. Exemplarysamples include blood, respiratory material, urine, CSF, spinal fluid,and other bodily fluids and tissue (such as soft tissue samples andwound material). Samples can contain a very low concentration of cellsand microorganisms, so low that direct from patient samples wouldtypically be deemed negative for the presence of microorganisms, despitea patient demonstrating symptoms consistent with an infection. Forexample, a sample may have a target bacterial concentration of as low asabout 10 cfu/mL or even 1 cfu/mL. The optical interrogation platform canbe integrated into a small (portable) incubator or contain a temperaturecontrolled environmental chamber to ensure normal bacterial growthduring the interrogation process.

System

FIG. 1 depicts a lens free imaging system using an optoelectronic sensorarray to generate a holographic image of sample objects. Large scaleoptical inferometry targets objects in a sample reaction chamber (e.g.,a flowcell, such as a microfluidic flow cell or perfusion chamber), withincident light. When light waves encounter an object—such as a microbialcell or debris—the light waves are distorted from their original pathand the interference or light scatter is recorded by the novel opticalsystem as a hologram. When an interference wave spot changes over aperiod of time, the system records that perturbation as a growingobject. Thus, in a phenotypic assessment of whether a viable cell ormicroorganism exists in a sample, having multiple sensors to screen arelatively large volume of sample in a short period of time may permitthe detection of cells in as little as 1.5 to 2 hours by capturingimages 15-30 minutes apart (or faster in some examples) over thatperiod. In principle, microbial cells can be detected within 2-3doubling times using this process.

An embodiment of the optical interrogation platform includes an in-lineholographic setup that includes a single-aperture multi-wavelengthillumination and a complementary metal oxide semiconductor (CMOS) sensorhaving a pixel pitch of 1.12 micrometers. Holograms obtained using theoptical interrogation platform are reconstructed—propagated viadiffraction theory, then intensity and phase retrieved, for exampleusing the iterative phase retrieval algorithm (such as Gerchberg-Saxton(GS)). A reference wave (illumination) can interact with sample aspropagating thru sample and at any point along reference wave, and everypoint becomes another point source (Huygens), and sensor recordsinterference pattern of all of these waves (e.g., hologram). Because asensor records only the intensity component of the complex wave function(hologram), phase component needs to be extracted. To gain back phase,it can be reconstructed numerically. Step 1 goes back to complexdiffraction pattern in a particular focal plane via diffractiontheory—solving Fresnel-Kirchoff integral (using Fresnel approximation orconvolutional methods). Step 2 then reconstructs phase via iterativephase retrieval algorithm (such as GS). This platform can be paired withor contain a subsystem which, for certain types of samples (such aswhole blood and respiratory samples) performs necessary preparatorysteps, including but not limited to dilution, centrifugation,application of an electrical field, and spin-and-resuspension, to reduceamounts of non-bacterial debris load. The optical interrogation platformis scalable in space by “mosaicking” illumination-sensor “pairs” (e.g.,multiplexing), thereby providing extensible spatial configurations.

The in-line holographic configuration of a lens-free setup includesmulti-wavelength illumination to remove twin-distortion during the phaseretrieval stage as well as to improve resolution, but it could be anyin-line holographic setup such as multiple illumination apertures,single-wavelength or multi-wavelength illumination, and the like thatprovides effective resolution of ˜1 micro-meter/pixel. Although oneembodiment of the optical interrogation platform utilized a perfusionchamber mounted on top of a standard glass microscopy slide, theplatform may be designed to support imaging of other sample reactionchambers (e.g., flowcells, such as microfluidic channels or perfusionchambers) of a different configuration. FIG. 2A is a view of exemplarysample reaction chamber (e.g., perfusion chamber) mounted on glassmicroscope slides. FIG. 2B shows other exemplary perfusion chambers thatcan be used.

As previously noted, the optical interrogation platform automates growthdetection of microorganisms present in very low concentrations. Ingeneral, given the system's acquisition setup, each object suspended ina 3-dimensional (3D) volume has an optimal focal plane (plane of highestvariance) at any given time. The optimal focal plane can be foundautomatically for each object in the imaged volume for every time of thetime-lapse sequence. Then, tracking or equivalent spatial differencingtechniques can be employed to detect changes. Various optical transformsare possible to return to object. Because optical transforms underlyinghologram construction are linear operators, multiple holograms can beobtained and manipulate without loss of information, for example todetermine the presence or identification of a microbe, or measure growthof a microbe over time. FIG. 3 shows images obtained showing proof ofconcept of the optical interrogation platform using beads. The beadsmimicking bacteria, protozoa, or fungi in a patient sample can be “seen”using holograms, but are not visible by standard bright fieldmicroscopy. One hologram can be stored per 3D stack. The holographicimage of the volume may be stored, and then later the focal plane can bereconstructed numerically. The holograms may be stored as TIFF, JPEG, orother files routinely used in imaging.

In a “mosaicked” embodiment, the optical interrogation platform can beextended to conduct simple antimicrobial susceptibility testing. This isaccomplished by dedicating at least one microfluidic channel to servingas a growth control channel containing a sample in the absence ofantimicrobial agent. One or more other microfluidic channels containingsamples with antimicrobial agents added at appropriate concentrationsmay be utilized to assess antimicrobial susceptibility. For example,antibiotic susceptibility may be assessed by pre-mixing antibiotics withsample before introducing the mixture to one or more sample reactionchambers (e.g., flowcell, such as a microfluidic flowcell or perfusionchamber). Alternatively, antibiotics may be added after a sample hasbeen deposited into the sample reaction chamber, or antibiotics maydiffuse into contact with the sample from a dried-down state in thesample reaction chamber. During growth supporting conditions, microbialreplication in the “growth control” channel is compared to replicationin one or more antimicrobial channels over time can yield first-ordersusceptibility/resistance information.

Another embodiment of the optical interrogation platform supports amulti-channel scanning configuration (a tiled or “mosaicked”arrangement) can be extended with “growth control” channels that useselective media. Growth information from these channels, in conjunctionwith a standard “growth control” channel, can be used to infer bacterialfamilies and even species. In another embodiment, the opticalinterrogation platform permits microbial differentiation based onorganism morphology. Under certain optical resolution (˜0.5 um/pixel),the platform can be used to conduct morphological analysis todifferentiate morphology of individual bacterial cells within eachmicro-colony. Such information can be reported to a clinician.

In some embodiments, microorganism detection is achieved bysimultaneously scanning a sample volume as large as ˜300 microliters(μL) in a single optical field-of-view of up to 30 mm² of surface areaand up to 1 mm depth. The system can perform time-lapse imaging of thesame volume without mechanical motion at acquisition rates that are muchhigher than microbial division rates. Thus, the system enables theimaging of bacteria faster than a small number of their doubling events,such as fewer than 4 doubling events, fewer than 3 doubling events, orfewer than two doubling events. Some bacteria have a doubling time ofabout 15-30 minutes, meaning that detection of the presence of suchbacteria in a patient's sample could be achieved by the system in about30 to 45 minutes.

The detection of a microorganism in a sample in such a short period oftime permits clinicians to rapidly determine which patient samplesshould be further subjected to multiplexed automated single cell digitalmicroscopy. One such digital microscopy system is the fully automated,microscopy-based system disclosed in U.S. patent publication no. US2017/0023599 (herein incorporated by reference), which can performbacterial or yeast identification in about one (1) hour and AST in aboutfive (5) or fewer hours.

Methods of Identifying Microorganisms

The disclosed systems and devices can be used in methods to aid in thediagnosis of bacteremia and fungemia. They can also be used forsusceptibility testing of specific pathogenic bacteria commonlyassociated with or causing bacteremia. Results can be used inconjunction with other clinical and laboratory findings.

The disclosed methods can be used to quickly determine if the patienthas a microbial infection, and in some examples also identify themicrobes infecting the patient and identify which antimicrobial agentsare likely to be effective in treating the infection. Such methods arefaster than currently available assays. In currently available assays, apatient sample is incubated overnight in the presence of a culturemedium (such as at least 8 hours, at least 10 hours, at least 12 hours,or at least 18 hours, such as 8 to 24 hours or 8 to 12 hours), to allowfor microbes present in the sample to grow and multiply. If this resultsin a positive result (i.e., microbes are present), then additionalassays are used to identify the microbe, identify an effectiveantimicrobial agent to administer to the patient to treat theirinfection, and determine a minimal inhibitory concentration (MIC) ofantimicrobial agent to use. In contrast, in representative examples, thedisclosed methods and systems do not require overnight incubation of thepatient sample (e.g., in a culture medium) to determine whether thepatient sample is positive (i.e., microbes are present). In someembodiments, the disclosed methods identify the microbe(s) in thepatient sample (e.g., the genus, species, Gram status and/or strain ofthe microbe(s)) and identify an effective antimicrobial agent toadminister to the patient to treat their infection. In some examples,the disclosed methods take less than 3 hours to complete, such as lessthan 2 hours, less than 1.5 hours or about 1.5 hours, such as 1 to 3hours, or 1.5 to 2 hours. For example, using the disclosed methods, itcan take less than 3 hours, or less than 2 hours, such as 1.5 to 3hours, or 1.5 to 2 hours to determine if the sample is positive forbacteria, protozoa and/or fungi. For example, using the disclosedmethods, it can take less than 3 hours, such as less than 2 hours, suchas 2 to 3 hours, or 1.5 to 2 hours to identify the bacteria, protozoa,and/or fungi in the sample. For example, using the disclosed methods, itcan take less than 6 hours, less than 5 hours, or less than 4 hours,such as 3 to 6 hours, or 4 to 5 hours to identify the antimicrobial thatthe bacteria, protozoa, and/or fungi in the sample are sensitive to(e.g., will kill the bacteria, protozoa, and/or fungi).

Patients can include human and veterinary subjects, such as cats, dogs,cows, pigs, horses, sheep, goats, chickens, turkeys, and other birds,fish, and the like. In some examples, a patient is one who is known tohave or is suspected of having an infection (such as a bacterial orfungal infection). In one example, the patient is septic. Patientsamples include but are not limited to blood (e.g., whole blood, plasma,or serum), respiratory samples (such as bronchoalveolar lavage,oropharyngeal swab, nasopharyngeal swab, or sputum), saliva, urine,rectal swab, vaginal swab, tissue samples, or other biological specimens(such as those described herein).

In some examples, the patient sample contains only a single type ofmicroorganism. In other instances, the patient sample contains multipletypes of cells and microorganisms, such as mixtures of host cells,bacteria, protozoa, and/or fungi from differing genera, species, andeven strains (also known as “polymicrobial” samples), such as at least2, at least 3, at least 4 or at least 5 different types of bacteria,protozoa, and/or fungi. In some examples, the patient sample containsbacteria that are about 0.2 to 5 microns in width or diameter, such as0.5 to 5 microns in width or diameter, 1 to 2 microns in width ordiameter, or 0.5 to 1 microns in width or diameter. In some examples, apatient sample has a bacterial, protozoal, and/or fungal concentrationof less than 100 CFU/mL, less than 50 CFU/mL, or less than 10 CFU/mL,such as 1 to 20 cfu/ML, 1 to 100 CFU/mL, or 10 to 200 CFU/mL, such asabout 5 CFU/mL, 10 CFU/mL, about 20 CFU/mL, about 30 CFU/mL, about 40CFU/mL, about 50 CFU/mL, about 60 CFU/mL, about 70 CFU/mL, about 80CFU/mL, about CFU/mL, or about 100 CFU/mL. Thus, in some examples, themethod is capable of detecting bacteria, protozoa, and/or fungi at lessthan 100 CFU/mL, less than 50 CFU/mL, or less than 10 CFU/mL, such as 1to 20 cfu/ML, 1 to 100 CFU/mL, or 10 to 200 CFU/mL, such as about 5CFU/mL, 10 CFU/mL, about 20 CFU/mL, about 30 CFU/mL, about 40 CFU/mL,about 50 CFU/mL, about 60 CFU/mL, about 70 CFU/mL, about 80 CFU/mL,about CFU/mL, or about 100 CFU/mL.

In some examples, the patient sample is used directly. In otherexamples, the patient sample is subjected to one or more pre-processingsteps prior to imaging the sample. For example, the patient sample canbe concentrated, diluted, filtered, centrifuged, and/or separated beforeanalysis. In one example, the patient sample is lysed prior to analysis,for example to remove or reduce the number of non-bacterial ornon-fungal cells in the sample (e.g., to lyse blood cells). In someexamples, the patient sample is concentrated prior to analysis, forexample by centrifugation, which can also remove debris from the sample.In one example, the patient sample is subjected gel electrofiltration(GEF) (for example, to remove or reduce lysed cells and debris in thesample). GEF is a process of sample preparation that relies onapplication of an electrical field to cause sample debris present in asample to be separated from microorganism cells. Likewise, membraneassisted purification may be used in some embodiments, such that inresponse to an electrical potential, sample contaminants enter a porousfilter medium through one or more walls of a well disposed in the filtermedium, thereby separating them from cells of interest in the sample.

The patient sample (or portion thereof) is loaded in or introduced intoone or more solid supports (e.g., sample reaction chamber, such as aflowcell, microfluidic chancel, or perfusion chamber) of a samplecontainer that allows microbes to be visualized using the disclosedholographic methods. In one example, the support includes one or moreflowcells, microfluidic channels, perfusion chambers, or combinationsthereof, such as one on a microscope slide (or other solid support thatis optically transparent (e.g., glass or plastic) and, non-toxic tomicroorganisms). In some example, the perfusion chamber is a CoverWell′perfusion chamber (see FIGS. 2A and 2B). An exemplary perfusion chambermounted on a microscope slide is shown in FIG. 2A. The perfusion chamberhas a 20 mm diameter, with two ports (which allow for introduction ofthe sample, for example in a MHA gel suspension, as well as removal ofmaterials). In this example, the volume of the perfusion channel isabout 300 uL, with an effective imaging area of about 16 mm² at 0.9um/pixel. One skilled in the art will appreciate that other perfusionchambers can be used, such as other shapes (e.g., square, rectangular,oval, etc.). In addition, a single slide can include multiple individualsample reaction chambers, for example to allow multiple samples to beanalyzed contemporaneously, to allow a single sample to be analyzed inthe presence of different reagents (e.g., different growth media and/orantimicrobial agents), or combinations thereof (FIG. 2B).

After introducing the sample into a micro-fluidic channel, a perfusionchamber, or both (for example using a manual or automated pipettor), thecells in the sample can be immobilized, for example by entombing them inthree-dimensional space in a growth medium containing a gelling orsolidification agent, such as agar or agarose. In some embodiments, theentombing creates a microenvironment around the immobilizedmicroorganism, the characteristics of which are not influenced byneighboring microorganisms during the identification and/orsusceptibility testing periods. In some examples, the method includesretaining the microorganism on a detection surface of the support,thereby producing a retained microorganism, and subsequently introducinga gel medium (such as one containing agar) into the micro-fluidicchannel, perfusion chamber, or both, wherein the gel medium is incontact with the retained microorganism following introduction into themicro-fluidic channel, perfusion chamber, or both; immobilizing theretained microorganism in the micro-fluidic channel, perfusion chamber,or both at the same location where the microorganism is retained, toproduce an immobilized microorganism, wherein offspring of theimmobilized microorganism remain over time at a location with theimmobilized microorganism; and incubating the immobilized microorganismfor a period of time to allow for growth of the microorganism.

The sample and microorganisms therein can be incubated and immobilizedin the growth media in the sample reaction chamber at varioustemperatures, which in some examples is selected based on themicroorganism thought to be present in the sample. In some examples, theimmobilized microorganism are incubated at a temperature of at least 15°C., at least 20° C., at least 25° C., at least 30° C., or at least 37°C., such as 20° C. to 40° C., or 25° C. to 37° C.

The gel medium in which the microorganisms (and their offspring) areimmobilized in some examples does not include antimicrobial agents. Inone example, the gel medium in which the microorganisms (and theiroffspring) are immobilized is MHA, trypticase soy agar, or any othernon-selective culturing media, which permits growth of mostmicroorganisms. This can be referred to as the “growth control” channelor chamber. Thus, if microorganisms are present in the sample, theyshould grow and be detectable in this medium.

In some examples, the cells of the sample are present in liquid mediacontaining no gelling, porous or semi-solid agents and are notimmobilized. The system is used to identify and track specific cells andmicroorganisms.

For some samples, clinical decisions may be require an estimate of theconcentration of a pathogen (or multiple pathogens) in the sample, whichis usually reported on log-scale. For example, a clinician may determinethat a urine sample is negative if the concentration of a particularpathogen is less than 10⁴ cfu/mL. Therefore, treatment decisions may bemade based on such information. In some examples herein, reporting ofsuch information is allowed via direct optically resolved observation ofthe sample with accuracy that is better than half-log for each targetspecies in the sample (including polymicrobial samples). For example,urines are not typically pathogenic if less than 10⁴. Similarly,respiratory samples are not typically pathogenic if less than 10³. But,normal methods of quantifying such samples is very poor, with samplesplated and colonies counted, leading to highly error prone results, suchas merely obtaining second derivative of what was actually in thesample. In examples herein, time-evolved holographic results can be usedto directly discern particles from bacteria, which can producecost-effective and accurate quantity estimates.

In some examples, a sample is introduced into multiple sample reactionchambers (e.g., flowcell or perfusion chamber) of a sample container,such that at least one sample reaction chamber does not includeantimicrobial agents, and the others can include different antimicrobialagents, for example to assess antimicrobial susceptibility. In someexamples, the antimicrobial agents selected are based on theidentification of the microorganism(s) present in the sample. Forexample, antibiotic susceptibility may be assessed by pre-mixingantimicrobial agents with the patient sample before introducing themixture to one or more sample reaction chambers. Alternatively,antimicrobial agents may be added after a patient sample has beenintroduced into the sample reaction chambers, or antibiotics and/orantifungal agents may diffuse into contact with the patient sample inthe sample reaction chambers. During growth supporting conditions,microbial replication in the “growth control” channel is compared toreplication in one or more “antimicrobial channels” over time can yieldfirst-order susceptibility/resistance information. In some examples,different amounts of the same antimicrobial agent are used (e.g., serialdilution). In some example, the media containing the sample includes oneor more of the following antimicrobial agents: amikacin, ampicillin,ampicillin-sulbactam, aztreonam, ceftazidime, ceftaroline, cefazolin,cefepime, ceftriaxone, ciprofloxacin, colistin, daptomycin, oxycycline,erythromycin, ertapenem, gentamicin, imipenem, linezolid, meropenem,minocycline, piperacillin-tazobactam, trimethoprim-sulfamethoxazole,tobramycin, vancomycin, or combinations of two or more thereof. Otherantimicrobial agents that can be used also include aminoglycosides(including but not limited to kanamycin, neomycin, netilmicin,paromomycin, streptomycin, and spectinomycin), ansamycins (including butnot limited to rifaximin), carbapenems (including but not limited todoripenem), cephalosporins (including but not limited to cefadroxil,cefalotin, cephalexin, cefaclor, cefprozil, fecluroxime, cefixime,cefdinir, cefditoren, cefotaxime, cefpodoxime, ceftibuten, andceftobiprole), glycopeptides (including but not limited to teicoplanin,telavancin, dalbavancin, and oritavancin), lincosamides (including butnot limited to clindamycin and lincomycin), macrolides (including butnot limited to azithromycin, clarithromycin, dirithromycin,roxithromycin, telithromycin, and spiramycin), nitrofurans (includingbut not limited to furazolidone and nitrofurantoin), oxazolidinones(including but not limited to posizolid, radezolid, and torezolid),penicillins (including but not limited to amoxicillin, flucloxacillin,penicillin, amoxicillin/clavulanate, and ticarcillin/clavulanate),polypeptides (including but not limited to bacitracin and polymyxin B),quinolones (including but not limited to enoxacin, gatifloxacin,gemifloxacin, levofloxacin, lomefloxacin, moxifloxacin, naldixic acid,norfloxacin, trovafloxacin, grepafloxacin, sparfloxacin, andtemafloxacin), suflonamides (including but not limited to mafenide,sulfacetamide, sulfadiazine, sulfadimethoxine, sulfamethizole,sulfamethoxazole, sulfasalazine, and sulfisoxazole), tetracyclines(including but not limited to demeclocycline, doxycycline,oxytetracycline, and tetracycline), and others (including but notlimited to clofazimine, ethambutol, isoniazid, rifampicin, arsphenamine,chloramphenicol, fosfomycin, metronidazole, tigecycline, andtrimethoprim), or any combination of two or more thereof. Furtherantimicrobial agents include amphotericin B, ketoconazole, fluconazole,itraconazole, posaconazole, voriconazole, anidulafungin, caspofungin,micafungin, flucytosine, or any combination of two or more thereof.

In some examples, a sample is introduced into multiple sample reactionchambers of a sample container, such that at least one sample reactionchamber does not include antimicrobial agents, and the others caninclude different selective and differential growth media, for exampleto identify the microorganisms present in the sample. Some growth mediaonly supports growth and replication of particular microorganisms ortypes of microorganisms. Examples of selective and differential mediainclude blood agar, Eosin Methylene Blue (EMB) broth or EMB agar,mannitol salt broth or mannitol salt agar, MacConkey broth or MacConkeyagar, phenylethyl alcohol (PEA) broth or PEA agar, and YM broth or YMagar. For example, EMB broth or agar inhibits Gram-positive organisms,and is thus selective for Gram-negative species. MacConkey broth or agaris also selective for Gram-negative species and differential withrespect to lactose fermentation. Mannitol salt broth or agar (7.5% NaCl)is selective for staphylococci and differential with respect to mannitolfermentation, wherein fermentation of mannitol is only seen in thepathogenic species of Staphylococcus. PEA broth or agar is a selectivemedium which inhibits the growth of most Gram negative organisms. Forexample, MacConkey broth or agar can be used to select for Gram-negativebacteria (e.g., permits growth of Gram-negative bacteria), mannitol saltbroth or agar can be used to select for Gram-positive bacteria (such asStaphylococcus), and YM broth or agar can be used to select for yeast.Thus, detection of growth in a particular media, and in some examplesnot in other media, can allow for the identification of themicroorganism. For example, the identification of a microorganism, forexample determining its genus, species, Gram status and/or strain can beassessed by pre-mixing a particular growth media with the patient samplebefore introducing the mixture to one or more sample reaction chambers.Alternatively, particular growth media may be added after a patientsample has been introduced into the sample reaction chamber, orselective agents may diffuse into contact with the patient sample in thesample reaction chamber. During growth supporting conditions, microbialreplication in the “growth control” channel is compared to replicationin one or more “selective media” channels over time can yieldmicroorganism identification information. In some examples,alternatively or in addition to the use of “selective media” channels,the microorganisms are identified by morphology (e.g., shape, size)information obtained using the disclosed methods.

In some examples, the method includes determining the number of minimumnumber of microbes needed in the “growth control” channel to ensure thatall the channels containing the patient sample will have detectablemicrobes, if present in the sample. For example, serial dilutions can beperformed.

After the microorganisms are immobilized, they (and their offspring) areimaged using the disclosed holographic imaging methods. Images of one ormore (such as 1-100, for example, 2-25, 10-40, 30-80, or 50-100) fieldsof view (scaled depending on the volume of the channel to beinterrogated) of one or more microorganisms are captured. Multipleimages of the same field of view may be captured, for example under oneor more different imaging modalities. For example, images can beobtained over a period of seconds, to minutes, to hours, such as every 5minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, or 60 minutes.In some examples, images (such as images of the “growth control”channel) are obtained for at least 1 hour, at least 1.5 hours, at least2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least6 hours, at least 7 hours, at least 8 hours, at least 9 hours, or atleast 10 hours, such as 1 to 4 hours, 1 to 2 hours, 1.5 to 2 hours, or 2to 4 hours. The results from the “growth control” channel allow for thedetermination as to whether the patient sample contains bacteria,protozoa, and/or fungi, that is, whether the sample is “positive”.

In some examples, during a microbial identification assay period, imagesare obtained about every 5-30 minutes (such as about every 5 minutes, 10minutes, 15 minutes, 20 minutes, 25 minutes, or 30 minutes) for about 1to 8 hours, such as up to about 1.5 hours, 2 hours, 3 hours, 4 hours,4.5 hours, 5 hours, 6 hours, 7 hours, or 8 hours. In some examplesduring this stage, the images are subjected to morphological or otheranalysis (such as morphokinetic analysis) to identify characteristics ofthe imaged microorganisms, including one or more of noise, cross-talk,and microorganism morphology. The results from the “selective media”channel allow for the identification of the microorganisms (e.g., Gramstatus, genus, species, and/or strain) present in the patient sample.

In some examples, during an AST assay period, images are obtained aboutevery 5-30 minutes (such as about every 5 minutes, 10 minutes, 15minutes, 20 minutes, 25 minutes, or 30 minutes) for about 1 to 6 hours,such as about 1.5 hours, 2 hours, 3 hours, 4 hours, 4.5 hours, 5 hours,or 6 hours, creating a time-lapse record of microorganism growth. Duringthe AST process, various microorganism clone features can be measured,such as morphology and division rates and used for analysis. In someexamples, the growth of the microorganisms is measured qualitatively orquantitatively, for example by measuring the growth (or amount ofgrowth), lack of growth, or lysis of the microorganisms. Based on thebehavior of the microorganisms over time in the presence of the one ormore antimicrobials (for example, compared to a control that is notexposed to the antimicrobial(s)), a determination of susceptibility (orindeterminate susceptibility) or resistance of the identifiedmicroorganisms to each antimicrobial is made. The results from the“antimicrobial channel” allow for the determination as to whichantibiotic(s) the microorganism in the sample is susceptible to. Thus,in some embodiments, the system reports susceptibility, intermediate, orresistance to one or more antimicrobials. In some embodiments, thefollowing resistance phenotypes are reported by the system in responseto AST data analysis: Methicillin-resistant Staphylococcus aureus(MRSA), methicillin-resistant staphylococci (MRS), vancomycin-resistantS. aureus (VRSA), vancomycin-resistant Enterococcus species (VRE),high-level aminoglycoside resistance (HLAR) andmacrolide-lincosamide-streptogramin B resistance (MLSb). Upon thisdetermination, the subject from whom the sample was obtained can beadministered a therapeutically effective amount of the identifiedantibiotic(s).

Exemplary Microbes Detected

The disclosed methods and systems can be used to detect variousGram-positive and Gram-negative bacteria, protozoa, and fungi (e.g.,yeasts), including but not limited to: Staphylococcus aureus,Staphylococcus lugdunensis, coagulase-negative Staphylococcus species(Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcushominis, Staphylococcus capitis, not differentiated), Enterococcusfaecalis, Enterococcus faecium (Enterococcus faecium and otherEnterococcus spp., not differentiated, excluding Enterococcus faecalis),Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcusagalactiae, Streptococcus spp., (Streptococcus mitis, Streptococcuspyogenes, Streptococcus gallolyticus, Streptococcus agalactiae,Streptococcus pneumoniae, not differentiated), Pseudomonas aeruginosa,Acinetobacter baumannii, Klebsiella spp. (Klebsiella pneumoniae,Klebsiella oxytoca, not differentiated), Escherichia coli, Enterobacterspp. (Enterobacter cloacae, Enterobacter aerogenes, not differentiated),Proteus spp. (Proteus mirabilis, Proteus vulgaris, not differentiated),Citrobacter spp. (Citrobacter freundii, Citrobacter koseri, notdifferentiated), Serratia marcescens, Candida albicans, and Candidaglabrata.

Other specific bacteria that can be detected with the disclosed systemsand methods, include without limitation: Acinetobacter baumannii,Actinobacillus spp., Actinomycetes, Actinomyces spp. (such asActinomyces israelii and Actinomyces naeslundii), Aeromonas spp. (suchas Aeromonas hydrophila, Aeromonas veronii biovar sobria (Aeromonassobria), and Aeromonas caviae), Anaplasma phagocytophilum, Alcaligenesxylosoxidans, Actinobacillus actinomycetemcomitans, Bacillus spp. (suchas Bacillus anthracis, Bacillus cereus, Bacillus subtilis, Bacillusthuringiensis, and Bacillus stearothermophilus), Bacteroides spp. (suchas Bacteroides Bartonella spp. (such as Bartonella bacilliformis andBartonella henselae, Bifidobacterium spp., Bordetella spp. (such asBordetella pertussis, Bordetella parapertussis, and Bordetellabronchiseptica), Borrelia spp. (such as Borrelia recurrentis, andBorrelia burgdorferi), Brucella sp. (such as Brucella abortus, Brucellacanis, Brucella melintensis and Brucella suis), Burkholderia spp. (suchas Burkholderia pseudomallei and Burkholderia cepacia), Campylobacterspp. (such as Campylobacter jejuni, Campylobacter coli, Campylobacterlari and Campylobacter fetus), Capnocytophaga spp., Cardiobacteriumhominis, Chlamydia trachomatis, Chlamydophila pneumoniae, Chlamydophilapsittaci, Citrobacter spp. Coxiella burnetii, Corynebacterium spp. (suchas, Corynebacterium diphtheriae, Corynebacterium jeikeum andCorynebacterium), Clostridium spp. (such as Clostridium perfringens,Clostridium difficile, Clostridium botulinum and Clostridium tetani),Eikenella corrodens, Enterobacter spp. (such as Enterobacter aerogenes,Enterobacter agglomerans, Enterobacter cloacae and Escherichia coli,including opportunistic Escherichia coli, such as enterotoxigenic E.coli, enteroinvasive E. coli, enteropathogenic E. coli,enterohemorrhagic E. coli, enteroaggregative E. coli and uropathogenicE. coli) Enterococcus spp. (such as Enterococcus faecalis andEnterococcus faecium) Ehrlichia spp. (such as Ehrlichia chafeensia andEhrlichia canis), Erysipelothrix rhusiopathiae, Eubacterium spp.,Francisella tularensis, Fusobacterium nucleatum, Gardnerella vaginalis,Gemella morbillorum, Haemophilus spp. (such as Haemophilus influenzae,Haemophilus ducreyi, Haemophilus aegyptius, Haemophilus parainfluenzae,Haemophilus haemolyticus and Haemophilus parahaemolyticus, Helicobacterspp. (such as Helicobacter pylori, Helicobacter cinaedi and Helicobacterfennelliae), Kingella kingii, Klebsiella spp. (such as Klebsiellapneumoniae, Klebsiella granulomatis and Klebsiella oxytoca),Lactobacillus spp., Listeria monocytogenes, Leptospira interrogans,Legionella pneumophila, Leptospira interrogans, Peptostreptococcus spp.,Moraxella catarrhalis, Morganella spp., Mobiluncus spp., Micrococcusspp., Mycobacterium spp. (such as Mycobacterium leprae, Mycobacteriumtuberculosis, Mycobacterium intracellulare, Mycobacterium avium,Mycobacterium bovis, and Mycobacterium marinum), Mycoplasm spp. (such asMycoplasma pneumoniae, Mycoplasma hominis, and Mycoplasma genitalium),Nocardia spp. (such as Nocardia asteroides, Nocardia cyriacigeorgica andNocardia brasiliensis), Neisseria spp. (such as Neisseria gonorrhoeaeand Neisseria meningitidis), Pasteurella multocida, Plesiomonasshigelloides. Prevotella spp., Porphyromonas spp., Prevotellamelaninogenica, Proteus spp. (such as Proteus vulgaris and Proteusmirabilis), Providencia spp. (such as Providencia alcalifaciens,Providencia rettgeri and Providencia stuartii), Pseudomonas aeruginosa,Propionibacterium acnes, Rhodococcus equi, Rickettsia spp. (such asRickettsia rickettsii, Rickettsia akari and Rickettsia prowazekii,Orientia tsutsugamushi (formerly: Rickettsia tsutsugamushi) andRickettsia typhi), Rhodococcus spp., Serratia marcescens,Stenotrophomonas maltophilia, Salmonella spp. (such as Salmonellaenterica, Salmonella typhi, Salmonella paratyphi, Salmonellaenteritidis, Salmonella cholerasuis and Salmonella typhimurium),Serratia spp. (such as Serratia marcesans and Serratia liquifaciens),Shigella spp. (such as Shigella dysenteriae, Shigella flexneri, Shigellaboydii and Shigella sonnei), Staphylococcus spp. (such as Staphylococcusaureus, Staphylococcus epidermidis, Staphylococcus hemolyticus,Staphylococcus saprophyticus), Streptococcus spp. (such as Streptococcuspneumoniae (for example chloramphenicol-resistant serotype 4Streptococcus pneumoniae, spectinomycin-resistant serotype 6BStreptococcus pneumoniae, streptomycin-resistant serotype 9VStreptococcus pneumoniae, erythromycin-resistant serotype 14Streptococcus pneumoniae, optochin-resistant serotype 14 Streptococcuspneumoniae, rifampicin-resistant serotype 18C Streptococcus pneumoniae,tetracycline-resistant serotype 19F Streptococcus pneumoniae,penicillin-resistant serotype 19F Streptococcus pneumoniae, andtrimethoprim-resistant serotype 23F Streptococcus pneumoniae,chloramphenicol-resistant serotype 4 Streptococcus pneumoniae,spectinomycin-resistant serotype 6B Streptococcus pneumoniae,streptomycin-resistant serotype 9V Streptococcus pneumoniae,optochin-resistant serotype 14 Streptococcus pneumoniae,rifampicin-resistant serotype 18C Streptococcus pneumoniae,penicillin-resistant serotype 19F Streptococcus pneumoniae, ortrimethoprim-resistant serotype 23F Streptococcus pneumoniae),Streptococcus agalactiae, Streptococcus mutans, Streptococcus pyogenes,Group A streptococci, Streptococcus pyogenes, Group B streptococci,Streptococcus agalactiae, Group C streptococci, Streptococcus anginosus,Streptococcus equismilis, Group D streptococci, Streptococcus bovis,Group F streptococci, and Streptococcus anginosus Group G streptococci),Spirillum minus, Streptobacillus moniliformi, Treponema spp. (such asTreponema carateum, Treponema petenue, Treponema pallidum and Treponemaendemicum, Tropheryma whippelii, Ureaplasma urealyticum, Veillonellasp., Vibrio spp. (such as Vibrio cholerae, Vibrio parahemolyticus,Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrioalginolyticus, Vibrio mimicus, Vibrio hollisae, Vibrio fluvialis, Vibriometchnikovii, Vibrio damsela and Vibrio furnisii), Yersinia spp. (suchas Yersinia enterocolitica, Yersinia pestis, and Yersiniapseudotuberculosis) and Xanthomonas maltophilia among others.

Exemplary fungi that can be detected with the disclosed systems andmethods, include without limitation: Candida spp. (such as Candidaalbicans, Candida glabrata, Candida tropicalis, Candida parapsilosis,and Candida krusei), Aspergillus spp. (such as Aspergillus fumigatous,Aspergillus flavus, Aspergillus clavatus), Cryptococcous spp. (such asCryptococcus neoformans, Cryptococcus gattii, Cryptococcus laurentii,and Cryptococcus albidus), Fusarium spp. (such as Fusarium oxysporum,Fusarium solani, Fusarium verticillioides, and Fusarium proliferatum),Rhizopus oryzae, Penicillium marneffei, Coccidiodes immitis, andBlastomyces dermatitidis.

Exemplary protozoa include, that can be detected with the disclosedsystems and methods, include without limitation: Plasmodium (e.g.,Plasmodium falciparum), Leishmania, Acanthamoeba, Giardia, Entamoeba,Cryptosporidium, Isospora, Balantidium, Trichomonas, Trypanosoma (e.g.,Trypanosoma brucei), Naegleria, and Toxoplasma.

Example

A micro-fluidic channel was constructed by placing a cover well on topof a glass microscopy slide measuring about 20 mm in diameter and 1 mmin height. A patient sample was simulated by diluting an E. coli 25922isolate into Mueller-Hinton agar suspension. The concentration ofbacterial isolate was chosen such that there were approximately 10²bacteria per mL. The bacterial-agar suspension was premixed and pipettedinto an inlet opening on top of the cover well. Thereafter, thebacterial-agar suspension was subjected to a phase change to solidifythe agar and suspend the bacteria in three-dimensional space. Prior tothe beginning of image acquisition, the micro-fluidic channel was placedinside an incubator for lhr to promote the growth phase of the suspendedbacteria.

Time-lapse imaging was conducted on a laboratory benchtop at ambienttemperature (approximately 20° C.). Holograms of the full field-of-view(approximately 16 mm²) were acquired automatically every 30 minutes.Visible division of bacterial micro colonies were detected as early as60 minutes after the start of image acquisition. Reliably detectabledivision across most micro colonies in the suspension is achievedapproximately 2-3 hours after the start of acquisition for thesebacteria. Because detection is based on change over time, presence ofdebris is not expected to have a significant impact on time-to-detectionsensitivity. FIG. 4 shows images obtained by the optical interrogationplatform imaging E. coli growth over a period of 0 to 180 minutes. FIG.5 shows images obtained by the optical interrogation platform imaging E.coli growth during a period from 240 to 540 minutes.

Time-to-detection highly depends on the optical resolution supported bythe system. It is also related to growth media as the experiment usedagar phase changed to a gel to contain growth to a particularthree-dimensional location in the volume. Hence, tracking of individualmicro colonies was not necessary. Thus, the optical interrogation systemcan be used to detect the presence of growing microorganisms in abiological sample long before traditional methods are capable of doingso. Upon detection of a microorganism present at a very lowconcentration in a biological sample, the sample may be further testedto determine the identity of the microorganism and its susceptibility toantimicrobial agents.

Additional System Examples

In FIG. 6, an in-line holographic apparatus 600 is situated to determinethe presence of microorganisms 602, 604 immobilized or that are free tomove in a sample volume 606 of a biological sample container 607. Inrepresentative examples, the apparatus 600 can detect a variation overtime of an in-line hologram 608 of the sample volume 606, including inan automated fashion, through detection of the sample volume atpredetermined times, e.g., during incubation. For example, growth of themicroorganisms 602, 604 can produce variation of respective holographicinterference patterns 610, 612 of the in-line hologram 608. Variationsindicative of the presence of microorganisms 602, 604 can be detectedbased on time durations associated with microorganism growth rates, suchas doubling events, negative growth rates (e.g., rates associated withantimicrobial activity), etc. In typical examples, time durations ofdoubling events of the immobilized microorganisms 602, 604 are longerthan temporal resolutions typically associated with in-line holography,allowing some example systems to provide improved detection, detectionover time, imaging, and imaging over time capabilities and lower costswith simpler components and reduced storage and/or processingrequirements. In some examples, the in-line hologram 608 can be detectedand recorded at rates suitable for detecting doubling events, such as atleast twice the doubling rate, or faster, with substantially fasterrates possible depending on the detection requirements, such asmorphological detection, etc.

In some examples, the in-line holographic apparatus 600 includes areference beam source 614 situated to direct a reference beam 616 to thesample volume 606, a sample receptacle 618 situated to hold the samplevolume 606 in view of the reference beam 616, an optical sensor 620situated to detect the in-line hologram 608 formed by the reference beam616 and the sample volume 606, and a holography controller 622 coupledto the optical sensor 620 and configured to determine the variation overtime of the in-line hologram 608. The sample volume 606 can include oneor more (e.g., a plurality of) sample volume portions corresponding tovolumes in microfluidic channels, flow channels, perfusion chambers,etc., of the biological sample container 607 and that can containbiological samples, such as suspended biological samples withmicroorganisms to be detected, including immobilized microorganisms. Intypical examples, the sample receptacle 618 can include a tray or otherholding support that receives the biological sample container 607 suchthat the biological sample can be removable inserted into the in-lineholographic apparatus and held by the sample receptacle 618 so that thesample volume 606 can be imaged by the in-line holography apparatus 600.In some examples, the microfluidic channels, flow channels, perfusionchambers, or other parts of the biological sample container 607, canform at least part of the sample receptacle 618.

In representative embodiments, the reference beam source 614 includes apinhole aperture 624 situated to receive an illumination 628 from anillumination source 626 and the reference beam 616 is directed lens-freefrom the pinhole aperture 624 to the sample volume 606 and the opticalsensor 620. In some examples, the illumination source 626 includes oneor more light emitting diodes, laser, or other light source that issituated to produce the illumination 628 with multiple wavelengths thatcan be used to reduce a twin-image in the hologram 608. In some examplesthe illumination 628 and the reference beam 616 are incoherent, theillumination 628 and the reference beam 616 are coherent, or theillumination 628 is incoherent and the reference beam 616 is coherent.The shape, diameter, and shape quality (e.g., roughness, ellipticity,etc.) of the pinhole aperture 624 can vary in different embodiments. Intypical examples, the pinhole aperture is circular and has a diameterselected in range of 1 μm or smaller, 1 to 10 μm, 10 to 50 μm, 50 to 100μm, or larger, and together with the wavelength or wavelengths of theillumination 628 determines the numerical aperture of the reference beam606.

In typical examples, the reference beam 616 diverges to define animaging area 630 and field of view of the sample volume 606 based on thedivergence angle of the reference beam 616 and the distance between aposition Z_(APERTURE) of the pinhole aperture 624 and top and bottomplane positions Z_(VOL1), Z_(VOL2) of the sample volume 606. Inrepresentative embodiments, the positions Z_(VOL1), Z_(VOL2) aresufficiently proximate each other, i.e., the sample volume 606 issufficiently thin, in relation to the distance between Z_(VOL1) andZ_(APERTURE) that the positions Z_(VOL1), Z_(VOL2) can be consideredeffectively one position for purposes of the imaging area 630. Inrepresentative examples, the distance Z_(APERTURE)-Z_(VOL1) is selectedto be in the range of 40 mm to 100 mm, though distances smaller than 40mm or greater than 100 mm are also possible. In some examples, thethickness of the sample volume 606 corresponding to the differenceZ_(VOL1)-Z_(VOL2) is 2 mm or smaller, 1 mm or smaller, 0.5 mm smaller,etc. Representative imaging areas of the sample volume 606 for a singleaperture and reference beam can vary, and can include 50 mm² or larger,40 mm² or larger, 30 mm² or larger, 20 mm² or larger, 10 mm² or larger,5 mm² or larger, or smaller than 5 mm², by way of example. In someexamples, areas are increased with additional apertures and/or opticalsensors. Imaging areas for a single field of view can typicallycorrespond to large volumes, including greater than 2 μL, 5 μL, 10 μL,20 μL, 50 μL, or greater.

Representative examples of the optical sensor 620 include CMOS or CCDtype sensors, that include a plurality of pixels 632 (shown in anexpanded cutout) arranged with one or more pixel pitches A to form asensor surface 634 situated to detect the in-line hologram 608. Inrepresentative examples, the pitch A corresponds to a detectorresolution that is sufficiently small to detect the spatial intensityvariation of the holographic interference patterns 610, 612 of thein-line hologram 608 or to detect a variation over time of the spatialintensity variation, such as a pitch A of 10 μm/pixel, 5 μm/pixel, 2μm/pixel, 1 μm/pixel, or smaller. In a particular embodiment the pitch Ais 1.12 μm/pixel. In some examples, the pixel pitch A is selected to besufficiently small to detect spatial intensity characteristics of thein-line hologram 608 that are associated with morphologicalcharacteristics of the microorganisms 602, 604. In some examples,characteristics of the reference beam 616 or other components of thein-line holographic apparatus 600 are varied to enhance detectionresolution, including varying reference beam wavelength to sampledifferent portions of the pixels 632, varying aperture characteristicssuch as aperture angles, super-resolution techniques employing relativesuperposition of sample and illumination, and numerical techniques suchas super-resolution via compressed sensing.

In representative examples, the distance between the bottom planeposition Z_(VOL2) of the sample volume 606 and the plane positionZ_(HOLO) of the sensor surface 634 is 10 mm or smaller, 5 mm or smaller,2 mm or smaller, etc. In a particular example, the distanceZ_(VOL2)-Z_(HOLO) is 4 mm or smaller. In some examples, the distanceZ_(VOL2)-Z_(HOLO) is selected so as to provide suitable spatialcharacteristics for the interference patterns 610, 612 or otherinterference characteristics of the in-line hologram 608, such as asufficient propagation distance to produce a corresponding holographicinterference between the reference beam 616 and object scattered beams611, 613. While some examples of the sample volume 606 are generallydepicted with a cuboid shape, other shapes can be used, includingcylindrical, frustum, elliptoid, etc.

The holography controller 622 includes a detector control 640 and anillumination control 642 respectively in communication with the opticalsensor 620 and the illumination source 626 or other light modulationdevice, such as an optical chopper, light modulator, etc., so that theillumination 628 is provided to form the reference beam 616 andassociated hologram 608 that is detected by the optical sensor 620 andso that the optical sensor 620 is ready (e.g., gated, reset, etc.) todetect the hologram 608.

In some examples, the holography controller 622 is a computing devicethat includes a memory 636 that can include one or more computerreadable instructions, such as program modules, that can be executed byat least one processor 638, such as one or more of a microcontrollerunit, complex programmable logic device, field programmable gate array,application-specific integrated circuit, programmable logic controller,computer system, etc., arranged singularly or in distributed fashion.Generally, program modules include routines, programs, objects,components, data structures, etc., that perform particular tasks orimplement particular abstract data types. Moreover, the disclosedtechnology may be implemented with other computer system configurations,including hand-held devices, multiprocessor systems,microprocessor-based or programmable consumer electronics, network PCs,minicomputers, mainframe computers, etc.

The memory 636 can includes read only memory (ROM) and random accessmemory (RAM), one or more storage devices, such as a hard disk drive forreading from and writing to a hard disk, a magnetic disk drive forreading from or writing to a removable magnetic disk, and an opticaldisk drive for reading from or writing to a removable optical disk (suchas a CD-ROM or other optical media). The drives and their associatedcomputer-readable media provide nonvolatile storage of computer-readableinstructions, data structures, program modules, and other data for theholography controller 622. Other types of computer-readable media whichcan store data that is accessible by a PC, such as magnetic cassettes,flash memory cards, digital video disks, CDs, DVDs, RAMs, ROMs, etc.,may also be used in the example holographic control environment.

In some examples, a number of program modules can be stored in thememory 636, including an operating system, one or more applicationprograms, other program modules, and program data. In further examples,a user can enter commands and information into the holography controller622 through one or more input devices, such as a keyboard, and apointing device, such as a mouse. Other input devices can be included.Thus, in representative examples, the various routines, programs, andprogram modules can be automated so that biological samples may bereceived by the in-line holography apparatus 600 so that tests can beperformed on the biological samples with little intervention from auser. In some examples, a display device 648 is situated to displayimages of the hologram 608 or holographic reconstructions of one or moreplanes of the sample volume 606, including time-lapse images or videorecordings associated with microorganism growth or size variation.

An image timer 644 can be used in different examples to synchronizedetection and recording of the hologram 608 or associated holograminformation in the memory 636 for subsequent comparison or imaging. Insome examples, the holography controller 622 includes a spatialdifference comparison routine 646 that determines spatial differencesassociated with holograms recorded at different times, such as bycomparing variations of holographic fringes and other spatial frequencyencoding features. In some examples, spatial differences can bedetermined between hologram reconstructions of one or more planes of thesample volume 606 associated with holograms recorded at different times,including area and texture variations of one or more objects, such asthe microorganisms 602, 604. Other approaches may include “learning”holographic representation of growth over time with higher-dimensionaltechniques such as Convolutional Neural Networks and conducting directinference on observed pixels at each time point of the time-lapse. Inrepresentative examples, improved microorganism detectability isachieved for the sample volume 606 based on the immobilized but growing(or declining) microorganisms and background immobilized objects thathave spatial characteristics that do not vary over time. For example, incomparing spatial differences, a substantial set of the backgroundobjects and associated signal characteristics can be eliminated throughimage subtraction so as to improve a signal to noise ratio for thespatial difference comparison routine 646.

In some examples, the holography controller 622 includes a numericalreconstruction routine 652 that is configured to reconstruct one or moreplanes of the sample volume 606 associated with the microorganisms 602,604 based on the hologram 608 detected at the plane Z_(HOLO) of theoptical sensor 620. In general, such routines approximate solutions toFresnel-Kirchoff diffraction integral by employing a Fresnelapproximation (Fresnel integral) or a convolutional approach at anyfocal plane between Z_(VOL1) and Z_(VOL2), followed by intensity andphase extraction. In some examples, the numerical reconstruction routine652 includes a Gerchberg-Saxton algorithm. Objects, such as themicroorganisms 602, 604, that are identified can be tracked in an objectgrowth table 654 for comparison with holograms detected at later timesto determine if a spatial variation occurs that is associated with thepresence of the microorganisms 602, 604.

The disclosed technology may also be practiced in distributed computingenvironments where tasks are performed by remote processing devices thatare linked through a communications network. In a distributed computingenvironment, program modules may be located in both local and remotememory storage devices. For example, holographic comparison by theholography controller 622 associated with an indication as to thepresence of the microorganisms 602, 604, immobilized or motile, in thesample volume 606 can be performed locally upon receiving a plurality ofholograms for comparison or can also be performed remotely in spaceand/or time from the detection of holograms by the optical sensor 620.In some examples, the holography controller includes a networkcommunication connections 650 to communicate with external device orother computers, e.g., through a local area network (LAN) or wide areanetwork (WAN). The in-line holographic apparatus 600 can further includea movement stage 652 coupled to the sample volume 606, such as through aside of the sample receptacle 618, though it will be appreciated thatvarious couplings can be used to provide translational and/or rotationalmovement of the sample volume 606. The controller 622 can include astage control 654 that can command and cause movement of the samplereceptacle 618 to different positions, e.g., based on a flow cell map656, so that different flow cells or portions of a flow cell can bealigned in view of the reference beam 616 and interrogated, e.g.,between the reference beam source 614 and the optical sensor 620. Inrepresentative examples, a movement stage 652 can be omitted.

FIG. 7 shows an example in-line holographic apparatus 700 that has amosaicked field of view 702 of a sample volume 704 with a plurality ofreference beams 706 a-706 d emitted from respective pinhole apertures708 a-708 d based on respective illuminations 710 a-710 d (typicallymulti-wavelength) received from respective illumination sources 712a-712 d. In some examples, a single illumination source can be used toilluminate the pinhole apertures 708 a-708 d, and in other examplesother quantities of illumination sources can be used. In particularexamples, a pinhole aperture 708 a can include a plurality of spacedapart pinhole apertures, typically at a small distance (e.g., less thanabout 1 mm), and the illumination source 708 a can include separateillumination sub-sources emitting at separate respective wavelengths andcoupled to the respective spaced apart pinhole apertures. Alternatively,each spaced apart pinhole aperture can be coupled to a respectivewavelength filter so that reference subbeams at different wavelengthsare emitted from the respective spaced apart pinhole apertures. Theother pinhole apertures 708 b-708 d and illumination sources 712 b-712 dcan be similarly configured and detected holograms can be registeredwith respect to each other based on subsampling of the respectiveoptical sensor portions 718 a-718 d.

The sample volume 704 typically includes a suspended biological samplehaving immobilized or motile microorganisms 714 a-714 d in respectivesample volume portions 715 a-715 d. Sample containers and samplereceptacles are omitted for clarity and convenience of illustrationthough it will be appreciated that various containers and receptaclesfor supporting and manipulating biological samples can be used.Respective in-line holograms 716 a-716 d are formed and detected,including holographic pattern features 720 a-720 d that are generatedbased on the immobilized or mobile microorganisms 714 a-714 d, with thedifferent optical sensor portions 718 a-718 d. In some examples, theoptical sensor portions 718 a-718 d can form a single sensor or multiplesensors. In some mosaic embodiments, at least one of the sample volumeportions 715 a-715 d is used as a growth control and one or more othersof the sample volume portions 715 a-715 d include selective media orantimicrobial agents. In further mosaic embodiments, the sample volumeportions 715 a-715 d are not isolated from each other and the multiplereference beams 706 a-706 d effectively increase the imaging area of thein-line holographic apparatus 700. In some examples, the reference beams706 a-706 d have respective imaging areas that can overlap at the samplevolume 704 so that the mosaicked field of view 702 can have continuouscoverage over at least a portion of the sample volume 704 including allof the sample volume 704 in selected examples. The sample volume can berelatively large, with some examples have a volume of 0.01 mL orgreater, 0.05 mL or greater, 0.1 mL or greater, 0.5 mL or greater, or 1mL or greater, etc.

The in-line holographic apparatus 700 can include a holographycontroller 722 that can control holographic imaging and holographicimaging over time of the sample volume 704. The holography controllertypically includes at least one processor 724, and a memory 726 thatincludes stored instructions associated with the detection of theholograms 716 a-716 d. In representative examples, the holographycontroller 722 includes an illumination control 728 that can cause theillumination sources 712 a-712 d to generate the illuminations 710 a-710d at respective times or periods that can be the same or different fromeach other, and can be controlled based on an image timer 732. Inrepresentative examples, the hologram controller 722 includes a detectorcontrol 730 in communication with the optical sensor portions 718 a-718d so as to receive one or more hologram signals associated with theholograms 716 a-716 d. In some examples, objects are detected, such asthe microorganisms 714 a-714 d, and monitored over time, e.g., in anobject growth table 734, so as to determine the presence of themicroorganisms 714 a-714 d, or other characteristics, such asmorphological characteristics, microorganism quantity or concentration,growth control characteristics, antimicrobial responsiveness, selectivemedia based species determination, etc., depending on the particularapplication. In some examples, objects and object variations (e.g.,growth), can be determined with a spatial differences comparison routine738 that compares spatial variations within the holograms 716 a-716 d,within reconstructions of the sample volume portions 715 a-715 d basedon the holograms 716 a-716 d and one or more reconstruction algorithms736, or spatial variations over time of holograms or reconstructions. Insome examples, one or more displays are included to show holographicinformation, amplitude and/or phase features, reconstructed samplevolume features, sample volume feature variation over time (e.g.,microorganism growth or decline), etc. Some examples can include one ormore communications modules for remote communication. Selected examplescan include a movement stage (not shown) to move the sample volume.

FIG. 8 depicts an example method 800 for detecting the presence of amicroorganism. At 804, a first in-line hologram of a sample volume isdetected at a first time, and at 812, a second in-line hologram of thesample volume is detected at a second time. At 818, a variation overtime associated with the in-line holograms is determined (e.g., betweenthe first and second in-line holograms) that is associated with anindication that one or more objects immobilized in the sample volume isa microorganism. In some examples, at 802, the sample volume includes abiological sample that includes microorganisms suspended in a porousmedium so as to immobilize the microorganisms to be detected. In someexamples, at 806, the spatial characteristics of objects in the samplevolume are reconstructed from the first in-line hologram, forming afirst reconstruction of the sample volume.

Reconstructions can be performed according to various methods, such aswith various diffraction propagation approximations (e.g., Fresnelapproximation) and iterative phase retrieval approaches, such asGerchberg-Saxton algorithms. The in-line holograms are generated as areference beam interacts with the sample volume and produces a complexinterference pattern based on object beams that are formed from opticalinteraction between the reference beam and the immobilized objects andresulting interference between the reference beam and object beams.Phase components associated with the immobilized objects is extractedfrom the intensity characteristics of the hologram. In typical examples,a Fresnel integral is applied to the hologram intensity to determine aplane associated with an immobilized object and an iterativeGerchberg-Saxton algorithm is used to reconstruct intensity and phase ofthe immobilized object.

In some examples, at 808, the reconstructions allow a determination ofthe position (e.g., a z-position, an x-y position, an x-y-z position,etc.) of one or more of the objects immobilized in the sample volumebased on spatial differences of the first in-line hologram or the firstreconstruction. In typical examples, at 810, the suspended biologicalsample is incubated in an environment conducive to microorganismreplication. At 814, in some examples, spatial characteristics of thesample volume are reconstructed from the second in-line hologramdetected at a later time, sometimes selected in relation to a suitablemicroorganism division rate or other biological rate. In representativeexamples, at 816, the first in-line hologram and the second in-linehologram, and/or the reconstructions of the first in-line hologram andthe second in-line hologram, are compared so as to identify holographicand/or reconstructed spatial differences, so that the variations overtime can be determined at 818. In some examples, growth detection isperformed without reconstructing the precise position and/or plane ofthe immobilized object, or without performing reconstruction at everyholographic detection event. Various examples herein can use thelinearity of optical transforms associated with reconstruction andmanipulate holographic information (e.g., add, subtract, etc.) withoutloss of information. Additionally, intensity variation of the hologramsover time (e.g., spatial intensity variation) can be used to determinemicroorganism presence. In typical examples, at 820, multiple hologramscan be obtained so that numerical features of the detected objects canbe accumulated over time. The accumulated features can be associatedwith an indication that one or more of the immobilized objectscorresponds to a microorganism in the suspended biological sample. Insome examples, at 822, the phenotypic behavior of an object can beclassified based on the accumulated numerical features, such as growth,death, lysis, filamentation, debris, etc.

FIG. 9 shows an example method 900 that includes, at 902, suspending abiological sample in a sample volume having a plurality of flow-cellvolumes isolated from each other such that at least one cell correspondsto a growth control and one or more other cells correspond toanti-microbial or selective media cells. At 904, a variation over timeof an in-line hologram of at least the growth control cell is detected.At 906, a correspondence between the detected variation and a presenceand/or concentration of a microorganism in the growth control cell isdetermined. At 908, a microorganism concentration sufficient to indicatea presence in the one or more other anti-microbial or selective mediacells is determined.

FIG. 10 is an example method 1000 that includes, at 1002, suspending abiological sample in a sample volume containing a growth mediumsupporting a microorganism of the biological sample therein. Inrepresentative examples, the supporting growth medium allows themicroorganism to move within the sample volume, though the microorganismcan also be immobilized by the supporting growth medium. At 1004, anautomated in-line holographic apparatus that typically directs anillumination beam lenslessly from a pin-hole aperture through the samplevolume to an optical detector, detects a first in-line hologram of thesample volume at an initial time (e.g., at a beginning of a test or at aselected time or sequence point during the test). At 1006, the 3Dspatial characteristics of the sample volume are reconstructed from thefirst in-line hologram, so as to form a first hologram reconstruction.Various techniques can be used for hologram reconstruction, diffractiontheory (e.g., iterative Gerchberg-Saxton), and/or deep learning (e.g.,convolutional neural networks). For example, in deep learningapproaches, such as convolutional neural networks, the network layerscan be supervised and the network activations can be trained to map rawhologram (interferometric) space into in-focus image plane at aspecified focal distance.

In selected examples, hologram reconstruction processes can includepre-processing of detected hologram data. For example, multi-wavelengthhologram registration can be used where multiple pinhole apertures arephysically separated by less than about 1 mm to form fixed predeterminedoffsets, such as with multiple wavelengths directed to a common samplevolume or sample volume portion through the respective proximateapertures. The pixel grid of the optical detector subsampled with themultiple wavelengths, and the acquired image data is shifted relative toeach other on an upsampled grid such that the relative offsets areeliminated. De-noising of the detected hologram data can be providedwith deep learning approaches (such as convolutional neural networks) ordeconvolution of an estimated/theoretical Point Spread Function (PSF) in2D or volumetric PSF in 3D. For example, de-noising with convolutionalneural networks can remove or suppress imaging sensor non-uniformities(e.g. pixel response non-uniformity or striping), an image degradationof the optical system (e.g. PSF), and diffraction ring cross-talkinterference, including without formulating analytical models forcorresponding sources of noise. In convolutional neural networkexamples, the network layers are typically trained using one or moresuboptimally acquired holograms (single-wavelength/single-aperture ornumerically degraded) where target data is a higher fidelity hologram(multi-wavelength/multi-aperture, multi-sampled/averaged). Thecorresponding trained network can then be applied to both lower-fidelityholograms as well as higher-fidelity holograms to suppress various noisecontributions, such as those described above. In some examples, in amanner similar to PSF deconvolution in optical (e.g., confocal)microscopy, PSF of a lens-free holographic system can be establishedeither empirically (e.g., by recording a signal associated withparticles below a resolution limit) or analytically. Techniques such asRichardson-Lucy algorithm can then be employed to deconvolve (or “takeout”) the PSF from the image data. The principal difference is that inlens-free imaging the above procedure can be applied directly to a rawhologram (i.e., before reconstruction). By pre-processing the detectedhologram data, the de-noising and/or deconvolution can improvevolumetric position estimation accuracy as well as amplitude and phaserepresentation of small spherical objects that approximate pointsources. Such approximations can be particularly applicable and validfor individual and/or clustered bacteria.

At 1008, from the sample volume reconstruction, a 3D position of one ormore objects in the sample volume (typically many objects in biologicalsample volumes) and/or morphological characteristics of the one or moreobjects in the sample volume can be determined, based on amplitude andphase characteristics associated with the first in-line hologram and/orfirst reconstruction. The suspended biological sample is typicallyincubated in an environment conducive to microorganism replication, at1010, for a predetermined time period. In representative examples,multiple holograms are detected in a test run at different points intime, and the time intervals need not be identical. Time resolution andtime interval variation can be selected based on incubationcharacteristics, growth media, microorganism growth stages, etc. At1012, a second in-line hologram is detected at a second time. The secondin-line hologram is reconstructed at 1014, and can use one or moretechniques that were used in the reconstructions of the first in-linehologram.

At 1016, a 3D position of one or more objects in the sample volumeand/or morphological characteristics of the one or more objects in thesample volume can be determined, based on amplitude and phasecharacteristics associated with the second in-line hologram and/orsecond reconstruction. In some examples, detected hologram data orrespective reconstructions can be compared over time to determine objectlocations and characteristics by analyzing differential variations on aspatial (or per-pixel) basis. Deep learning approaches based on Bayesianstatistical inference, including convolutional neural networks, can alsobe employed to recognize and quantify variation patterns arising fromdifferential holograms or differential reconstructed images. Inconvolutional neural network examples, the network is trained in asupervised fashion to recognize variational spatial patterns due to, byway of example, multiple species of bacteria and fungus versus otherbiological or non-biological particles.

Because the objects detected with the first hologram may grow, die,move, or provide other microorganism signatures that vary over the timeinterval between the first and second hologram detections, objectsdetected at 1016 may be closely related to objects detected in the firstin-line hologram. In some examples, at 1018, an object of the one ormore objects detected from the second in-line hologram and/or secondreconstruction is associated with an object of the one or more objectsdetected from the first in-line hologram and/or the firstreconstruction, based on proximity and/or morphological amplitude/phasecharacteristics. In some immobilized sample volume examples, objectassociations can be omitted or relaxed as the object does not changeposition (though growth, death, and/or other morphologicalcharacteristics may change) between first and second hologram detectionsdue to the immobilizing growth medium. At 1020, an object track for theassociated objects can be created over time to accumulate position,morphological, and amplitude/phase characteristics for the trackedobject. Object tracks can be 1D, 2D, and/or 3D in some examples. Inselected immobilized examples, object tracks can be omitted. In someexamples (and particularly convenient in immobilized examples),numerical features of a detected object can be accumulated over timethat are associated with an indication that the immobilized object is amicroorganism suspended in the biological sample volume. At 1022,phenotypical behavior of the tracked object can be classified, such asobject motility, growth, death, lysis, filamentation, debris, etc. At1024, additional objects can be associated, object tracks formed for theadditional tracked objects, and phenotypical behavior of the trackedobjects classified, individually or as a population. In selectedimmobilized examples, motility can be omitted. In either mobilized orimmobilized examples, based on the resolution of the apparatus (e.g.,resolving 10 μm, 5 μm, 1 μm, or 0.5 μm dimension of the sample volume,for relative large fields of view) and the ability to identifyindividual objects, actual object quantities and correspondingvolumetric concentrations in the sample volume can be determined,including individual cells or populations of cells.

FIG. 11 is an example iterative object association method 1100 in thetesting a sample volume that can contain a microorganism to be detected.At 1102, an in-line hologram is provided of time t_(i), a correspondinghologram reconstruction can be provided, and position and/or morphologyof objects at time t_(i) in the sample volume are provided that aredetermined based on the t_(i) in-line hologram and/or t_(i)reconstruction. For example, if t_(i) corresponds to a first in-linehologram of a series of holograms for a test of the sample volume, thenthe in-line hologram can be produced and detected at the time t_(i), thesample volume reconstructed, and/or object positions determined ratherthan, e.g., being provided in another way, such as through access from alocal or remote data storage. At 1104, a selected time intervalt_(i+1)-t₁ is provided after the time t_(i). At 1106, an in-linehologram is detected at time a corresponding hologram reconstruction isproduced, and position and/or morphology of objects at time t_(i+1) inthe sample volume are determined based on the t_(i+1) in-line hologramand/or t_(i+1) hologram reconstruction. The t_(i+1) objects with t_(i)objects are compared and associated at 1108 based on proximity to and/ormorphological characteristics to identify object types. At 1110, a checkis performed as to whether the in-line hologram imaging test of thesample is complete for the sample volume. If the test is not yetcomplete, the time t_(i+1) can be set to a time t_(i) and the process ofproviding an in-line hologram at 1102 (which can correspond to thein-line hologram provided in the previous step 1106) can be repeated. Inrepresentative examples, object associations can be updated, revised,including with new object associations, as subsequent holograms areobtained, analyzed, and compared with previous hologram, sequences ofholograms, and/or object association histories. Multiple objects in asample volume can be associated and identified and thereby quantified.Based on the size of the sample volume and the ability to quantify themultiple objects within the sample volume at different growth stages,precise object concentrations (including for different object types ortaxa) can be determined.

FIG. 12 is an example of an iterative phenotype classification method1200 that can be used in testing a biological sample in a sample volumewith an automated in-line holography apparatus. At 1202, an in-linehologram is provided of time t_(i), a corresponding hologramreconstruction can be provided, and position and/or morphology ofobjects at time t_(i) in the sample volume are provided that aredetermined based on the t_(i) in-line hologram and/or t_(i)reconstruction. For example, if t corresponds to a first in-linehologram of a series of holograms for a test of the sample volume, thenthe in-line hologram can be produced and detected at the time t_(i), thesample volume reconstructed, and/or object positions determined ratherthan, e.g., being provided in another way, such as through access from alocal or remote data storage. At 1204, a selected time intervalt_(i+1)-t_(i) is provided after the time t_(i). At 1206, an in-linehologram is detected at the time t_(i+1), a corresponding hologramreconstruction is produced, and position and/or morphology of objects attime t_(i+1) in the sample volume, including previously identified andassociated objects in the sample volume or previously associated objects(e.g., that move, grow, die, etc.), are determined based on the t_(i+1)in-line hologram and/or t_(i+1) hologram reconstruction. In typicalexamples, objects are identified and object associations are formedafter a plurality of holographic samples in a time sequence of the test.At 1208, an object history of an associated object (e.g., an object thatchanges position in the sample volume through flagellation, or abacterial growth, splitting, including individual or populations, etc.)can be updated, e.g., in computer memory, based on changes of detectedor computed object parameters, such as an object track (e.g., a movementpath, a centroid position change of a population, filamentationdirection, etc.) or morphological characteristics (e.g., shape,microorganism features, patterns, colors, size, etc.). In some examples,objects can be compared between times time t_(i+1) and objectassociations produced or updated, similar to as shown in the examplemethod 1100. At 1210, a check is performed as to whether the set ofhologram events is sufficient (e.g., sufficient number of events and/ora sufficient duration for incubation, etc.) to support a phenotypicclassification of the identified objects based on the object histories.If a sufficient set of in-line hologram events has not yet beencollected, the time t_(i+1) can be set to a time t_(i) and the processof providing an in-line hologram at 1202 (which can correspond to thein-line hologram provided in the previous step 1206) can be repeated. Ifthe set of events is sufficient, at 1214, phenotypic behavior of one ormore of the identified objects, individually or as a population, isclassified based on the accumulated object history for the object. Insome examples, such classifications can be updated, revised (includingbeing replaced), as additional holograms in a test sequence aredetected. Classification of numerical features that represent phenotypicbehavior over time for an identified object can be accomplished withvarious techniques, such as, but not limited to, regression,discriminant analysis, decision trees, and/or neural networks (e.g.convolutional neural networks). Classification categories can include(but are not limited to) object motility, growth, death, lysis,filamentation, and debris. Detected objects that exhibit response thatcan be representative of bacterial phenotypic response can be selectedfor further analysis along with their respective measured features. Suchanalyses can be performed on an individual object basis as well aspopulation basis.

FIG. 13 and the following discussion are intended to provide a brief,general description of an exemplary computing environment in which thedisclosed technology may be implemented. Although not required, thedisclosed technology is described in the general context ofcomputer-executable instructions, such as program modules, beingexecuted by a computing unit, dedicated processor, or other digitalprocessing system or programmable logic device. Generally, programmodules include routines, programs, objects, components, datastructures, etc., that perform particular tasks or implement particularabstract data types. Moreover, the disclosed technology may beimplemented with other computer system configurations, includinghand-held devices, multiprocessor systems, microprocessor-based orprogrammable consumer electronics, network PCs, minicomputers, mainframecomputers, dedicated processors, MCUs, PLCs, ASICs, FPGAs, CPLDs,systems on a chip, and the like. The disclosed technology may also bepracticed in distributed computing environments where tasks areperformed by remote processing devices that are linked through acommunications network. In a distributed computing environment, programmodules may be located in both local and remote memory storage devices.

With reference to FIG. 13, an exemplary system for implementing thedisclosed technology includes a computing device 1300 that includes oneor more processing units 1302, a memory 1304, and a system bus 1306 thatcouples various system components including the system memory 1304 tothe one or more processing units 1302. The system bus 1306 may be any ofseveral types of bus structures including a memory bus or memorycontroller, a peripheral bus, and a local bus using any of a variety ofbus architectures. The memory 1304 can include various types, includingvolatile memory (e.g., registers, cache, RAM), non-volatile memory(e.g., ROM, EEPROM, flash memory, etc.), or a combination of volatileand non-volatile memory. The memory 1304 is generally accessible by theprocessing unit 1302 and can store software in the formcomputer-executable instructions that can be executed by the one or moreprocessing units 1302 coupled to the memory 1304. In some examples,processing units can be configured based on RISC or CSIC architectures,and can include one or more general purpose central processing units,application specific integrated circuits, graphics or co-processingunits or other processors. In some examples, multiple core groupings ofcomputing components can be distributed among system modules, andvarious modules of software can be implemented separately.

The exemplary computing device 1300 further includes one or more storagedevices 1330 such as a hard disk drive for reading from and writing to ahard disk, a magnetic disk drive for reading from or writing to aremovable magnetic disk, and an optical disk drive for reading from orwriting to a removable optical disk (such as a CD-ROM or other opticalmedia). Such storage devices can be connected to the system bus 1306 bya hard disk drive interface, a magnetic disk drive interface, and anoptical drive interface, respectively. The drives and their associatedcomputer-readable media provide nonvolatile storage of computer-readableinstructions, data structures, program modules, and other data for thecomputing device 1300. Other types of non-transitory computer-readablemedia which can store data that is accessible by a PC, such as magneticcassettes, flash memory cards, digital video disks, CDs, DVDs, RAMs,ROMs, and the like, may also be used in the exemplary computingenvironment. The storage 1330 can be removable or non-removable and canbe used to store information in a non-transitory way and which can beaccessed within the computing environment.

As shown in FIG. 13, the computing device 1300 is coupled to an outputdevice I/O 1332 so that suitable output signals (e.g., digital controlvoltage and/or current signals) are provided to imaging devices 1340 ofan in-line holography generator 1342. The imaging devices 1340 typicallyinclude illumination sources generating light at one or more wavelengthsand pinhole apertures to receive the illumination and lenslessly directthe illumination to a sample volume 1346. A hologram is formed at ahologram detector 1344. Input device I/O 1334 is coupled to the bus 1306so that data signals and/or values corresponding to in-line hologramsdetected with the detector 1344 can be stored in the memory 1304 and/orstorage 1330 and/or processed with the processing unit 1302. In someexamples, a control stage 1348, such as a translation and/or rotationstage, can be coupled to the sample volume (and/or the detector 1344 andimaging device 1340) so that relative movement between the sample volume1346 and illumination/detection beams can be produced. The control stage1348 can provide a translation so that different cells for the samplevolume 1346, e.g., for large sample volumes, can be illuminated anddetected at different times.

In representative examples, the detected holograms are used toreconstruct the 3D physical characteristics of the sample volume 1346,so that immobilized or mobile objects in the sample volume 1346 (such asmicroorganisms) can be detected. Imaging and/or detection intervals,gating, synchronization, etc., can be stored in a memory 1310A alongwith various data tables for storing detected hologram data, manipulateddata (e.g., holographic reconstructions), and algorithms for analyzingdata. For example, identified/associated objects (e.g., a movingmicroorganism, a growing bacterial colony, etc.) and unassociated orstatic objects can be stored in objects tables 1310B. Hologramreconstruction algorithms, such as Gerchberg-Saxton (GS) and/or Bayesiandeep learning methods, can be stored in a memory 1310C. Objectidentification, object tracking, and/or morphological identificationalgorithms, such as convolutional neural networks, can be stored in amemory 1310D. As objects are tracked and associated morphologicalcharacteristics detected, histories of object characteristics can bestored in a memory 1310E. Phenotype classifications that can bedetermined based on the object tracks and morphological characteristicscan be stored in a memory 1310F.

A number of program modules (or data) may be stored in the storagedevices 1330 including an operating system, one or more applicationprograms, other program modules, and program data. A user may entercommands and information into the computing device 1300 through one ormore input devices such as a keyboard and a pointing device such as amouse. Various other input devices can be used as well. These and otherinput devices are often connected to the one or more processing units1302 through a serial port interface that is coupled to the system bus1306, but may be connected by other interfaces such as a parallel port,game port, or universal serial bus (USB). In representative examples,the various routines, programs, and program modules can be automated sothat biological samples may be received by the in-line holographygenerator 1342. The in-line holography generator 1342 can include or becoupled to the computing device 1300 so that tests can be performed onthe biological samples with little intervention from a user. A monitor1350 or other type of display device is also connected to the system bus1306 via an interface, such as a video adapter. The monitor 1350 can beused to display hologram images, reconstructed sample volume images in2D or 3D (e.g., perspective images, focal planes, z-planes, etc.), timelapse images of growth, images with static objects and/or debrissubtracted, etc. Some or all data and instructions can be communicatedwith a remote computer 1360 through communication connections 1355(e.g., wired, wireless, etc.) if desired.

FIGS. 14A-14C are sample volumes 1400A-1400C with contents that can bedetected over time through generation and detection of in-line hologramswith a holographic apparatus. In sample volume 1400A, an object 1402A isdetected at a time t₀, which can correspond to an initiation of anincubation and test of the sample volume 1400A or a time at a selectedpoint during the test. At a time t₁, an object is detected at adifferent position and the holographic apparatus can determine that theobject is associated with the object 1402A, such as through a movementto the new position. Different detected characteristics can beassociated with the movement, such as the lack of, or change in thecharacteristics of (e.g., image variation corresponding to aflagellation or movement wake), the object at the position detected attime t₀. The object 1402A can be detected at subsequent times t₂ and t₃and an object track 1404A can be formed. As shown in FIGS. 14A-14C, thevarious object tracks and morphological characteristics can be detectedin one, two, and/or three spatial dimensions.

The sample volume 1400B shows a growth of an object 1402B in a motile orimmobilizing support media. For example, at a time t₀ the object 1402Bcan be detected. At subsequent times t₁-t₃, a growth is detected such asthrough the change in position of an object boundary that corresponds toan area enlargement associated with the object 1402B. An object track1404B can also be identified, e.g., based on centroid calculations ormorphological characteristics of the object 1402B (e.g., color, opacity,shape, size, etc.). In the sample volume 1400C, an object 1402C isdetected at a time t₀ with no other objects detected in the surroundingvolume, or with some objects detected that can be later subtracted asnot corresponding to growing microorganisms. At a time t₁, multipleobjects are detected surrounding the object 1402C defining a growingobject boundary 1404C (e.g., with no objects detected outside the objectboundary 1404C). In some examples, each individual object can bedetected and movement can be tracked. At later times t₂-t₃, additionalobjects are detected indicative of growth of the initial object 1402Cand defining respective growing population boundaries 1406C, 1408C. Anobject 1410C detected at time t₁ can be associated with a movement alonga track 1412C to a new position at time t₂. Another object 1414Cdetected at time t₂ can be associated with a movement along a track1416C to a new position at time t₃. Track characteristics, includingdirectional changes, can be determined based on additional hologramsbetween selected time intervals, debris and/or wake detection, andmorphological characteristics including associations between size orshape and movement speed/distance.

FIG. 15 is an example multiplexed method 1500 of testing biologicalsamples. At 1502, a biological sample is suspended in a sample volumehaving a plurality of flow-cells or chambers isolated from each othersuch that at least one cell corresponds to a growth control and one ormore other chambers correspond to anti-microbial or selective mediacells. At 1504, a variation over time is detected in multiple in-lineholograms of at least the growth control cell and phenotypic behavior ofindividual objects and/or populations of objects in at least the growthcontrol cell is classified based on the detected hologram variation. Thehologram variation can be derived from several methods including, butnot limited to image reconstruction or by using pixel intensity tocalculate a statistical variability metric including, but not limited tostandard deviation, range, interquartile range and coefficient ofvariation. At 1506, a correspondence is determined between the detectedphenotypic behavior and a presence, concentration, and taxon of amicroorganism or multiple microorganisms in at least the growth controlflow cell. At 1508, a presence, taxon, and antibiogram of amicroorganism or multiple microorganisms is determined based on at leastone growth control, at least one selective media, and at least oneanti-microbial flow cell.

FIG. 16 is an example hologram reconstruction framework 1600 with aconvolutional neural network. The hologram reconstruction framework 1600typically includes a training phase 1602-1608 that refines theparameters of the convolutional neural network. At 1602, a set ofhologram training data is provided to a deeply supervised multi-layerconvolutional neural network. Training data typically includes a set ofholographic data having a known ground truth amplitude/phase spatialreconstruction for a sample volume. At 1604, the training hologram datais processed through the deeply supervised convolutional neural networkto produce a reconstruction of the sample volume based on the inputtraining hologram data. At 1606, the output hologram-basedreconstruction is compared with the ground truth representation of thesample volume, and at 1608, based on the detected errors, the non-linearactivations (e.g., softplus, ReLU, etc.) of one or more network layersof the convolutional neural network are updated by back-propagatingcomparison error through the convolutional neural network, e.g., viagradient descent. A testing phase 1610-1614 is used on field samplesafter the convolutional neural network is sufficiently trained. At 1610,data corresponding to an in-line holographic image of a biological testsample volume is provided from an imaging detector and/ormemory/storage. At 1612, the data is processed through the traineddeeply supervised convolutional neural network, and at 1614 areconstruction of the sample volume based on the hologram data isproduced.

FIG. 17 is an example micro-object identification/classificationframework 1700 with a convolutional neural network. The micro-objectidentification/classification framework 1700 typically includes atraining phase 1702-1708 that refines the parameters of theconvolutional neural network to converge on an improved output accuracyas additional training data sets are processed. At 1702, a set ofhologram training data is provided to a deeply supervised multi-layerconvolutional neural network. Training data typically includes a set ofhologram data and/or hologram reconstruction data having a known groundtruth object identification and/or object classification correspondencefor a sample volume that includes various objects. At 1704, the trainingdata is processed through the deeply supervised multi-layerconvolutional neural network to produce an output object identificationand/or object classification, such as an identification of objects,object morphologies, object movements, and phenotypic classifications.At 1706, output identification and/or classification is compared to theground truth associated with the training data. At 1708, activations ofone or more network layers are updated by back-propagation (e.g.,through gradient descent) of the comparison error through theconvolutional neural network. A testing phase 1710-1714 can be used onfield samples after the convolutional neural network is sufficientlytrained. At 1710, data corresponding to an in-line holographic image orreconstructed 3D spatial image of a biological test sample volume isprovided. At 1712, the data are processed through the trained deeplysupervised convolutional neural network, and at 1714 an objectidentification and/or classification is produced based on the hologramor reconstruction data.

In view of the many possible embodiments to which the principles of thedisclosure may be applied including, but not limited to growth andphenotypical discrimination of cells, cell units, microbes andmicroorganisms, it should be recognized that the illustrated embodimentsare only examples and should not be taken as limiting the scope of thedisclosed technology. Rather, the scope of the disclosed technology isdefined by the following claims. We therefore claim all that comeswithin the scope of these claims.

What is claimed is:
 1. A method for detecting a cell in a sample,comprising: a. detecting a first in-line hologram of at least one objectin the sample; b. measuring a variation over time of the in-linehologram; and c. determining the presence or absence of phenotypicalbehavior or event for the at least one object in the sample based on themeasured variation over time of the in-line hologram thereby detectingat least one cell in the sample.
 2. The method of claim 1, furthercomprising creating an association between the first in-line hologramand a second in-line hologram.
 3. The method of claim 1, furthercomprising creating an object track over time.
 4. The method of claim 1,further comprising immobilizing the at least one object in the sample.5. The method of claim 4, wherein immobilizing the at least one objectin the sample comprises allowing the at least one object in the sampleto settle onto a surface in a reaction chamber or suspending the atleast one object in the sample in a porous medium.
 6. The method ofclaim 5, further comprising subjecting the porous medium to a phasechange to immobilize the at least one object in the sample in athree-dimensional space.
 7. The method of claim 1, further comprisinginterrogating the sample in an automated optical interrogation systemusing one or more optoelectronic sensors to locate an optical focalplane for each at least one object in the sample
 8. The method of claim1, wherein determining the presence or absence of phenotypical behavioror event for the at least one object in the sample further comprisesclassifying the output of at least one deeply supervised convolutionalneural network.
 9. The method of claim 1, further comprising detecting asecond in-line hologram of at least one second object in the sample. 10.A method for detecting the presence of a living cell in a sample,comprising: a. detecting a first in-line hologram of at least one objectin the sample; b. detecting a second in-line hologram of at least oneobject in the sample; c. measuring a variation over time between thefirst and second in-line hologram; and d. associating the variation overtime with a living cell thereby detecting the presence of a living cellin the sample.
 11. The method of claim 10, wherein the variation overtime corresponds to cell growth or decline.
 12. The method of claim 10,further comprising producing an output corresponding to a reconstructionof the spatial characteristics of the at least one object in the samplebased on a trained set of network layers.
 13. The method of 10, furthercomprising producing an output corresponding to (i) identification, (ii)morphology, (iii) movement, and/or phenotypic classification of the atleast one object in the sample based on a trained set of network layers.14. The method of claim 10, further comprising using the first andsecond in-line holograms to calculate a statistical variability metricto determine variation of output over time.
 15. The method of claim 10,further comprising dividing the first and second in-line holograms intosubsections to identify a localized feature demonstrating phenotypicalchanges over time.
 16. The method of claim 10, further comprisingdetermining a focal plane corresponding to a place of highest variancein the sample that is associated with the at least one object.
 17. Themethod of claim 10, further comprising reconstructing spatialcharacteristics of the sample based on the detected first and secondin-line holograms and a numerical reconstruction algorithm.
 18. Themethod of claim 10, wherein the sample includes sample volume portionsthat are present in a plurality of respective chambers comprisingdifferential growth media.
 19. The method of claim 1, further comprisingdisplaying a time-lapse image associated with the sample at a timeresolution that is faster than a cell division rate.
 20. An automatedsystem comprising: a. an automated holographic optical apparatussituated to determine the phenotypical behavior of an object in a samplebased on a detected variation over time of a hologram of the sample; b.an output of at least one data calculation module, and a phenotypicalbehavior of the cell unit, wherein the phenotypical behavior of the cellunit is classified based on the detected variation.